High oleic acid oils

ABSTRACT

The present invention relates to extracted lipid with high levels, for example 90% to 95% by weight, oleic acid. The present invention also provides genetically modified plants, particularly oilseeds such as safflower, which can used to produce the lipid. Furthermore, provided are methods for genotyping and selecting plants which can be used to produce the lipid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/869,763, filed Apr. 24, 2013, now allowed, claiming priority ofAustralian Patent Application No. 2012903992, filed Sep. 11, 2012 andthe benefit of U.S. Provisional Application No. 61/638,447, filed Apr.25, 2012, the contents of each of which are hereby incorporated byreference in their entirety.

REFERENCE TO SEQUENCE LISTING

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“190514_84095-AZ_Sequence_Listing_CAS.txt,” which is 134 kilobytes insize, and which was created May 14, 2019 in the IBM-PC machine format,having an operating system compatibility with MS-Windows, which iscontained in the text file filed May 14, 2019 as part of thisapplication.

FIELD OF THE INVENTION

The present invention relates to extracted lipid with high levels, forexample 90% to 95% by weight, oleic acid. The present invention alsoprovides genetically modified plants, particularly oilseeds such assafflower, which can used to produce the lipid. Furthermore, providedare methods for genotyping and selecting plants which can be used toproduce the lipid.

BACKGROUND OF THE INVENTION

Plant oils are an important source of dietary fat for humans,representing about 25% of caloric intake in developed countries (Brounet al., 1999). World production of plant oils is at least about 110million tons per year, of which 86% is used for human consumption.Almost all of these oils are obtained from oilseed crops such assoybean, canola, safflower, sunflower, cottonseed and groundnut, orplantation trees such as palm, olive and coconut (Gunstone, 2001; OilWorld Annual, 2004). The growing scientific understanding and communityrecognition of the impact of the individual fatty acid components offood oils on various aspects of human health is motivating thedevelopment of modified vegetable oils that have improved nutritionalvalue while retaining the required functionality for various foodapplications. These modifications require knowledge about the metabolicpathways for plant fatty acid synthesis and genes encoding the enzymesfor these pathways (Liu et al., 2002a; Thelen and Ohlrogge, 2002).

Considerable attention is being given to the nutritional impact ofvarious fats and oils, in particular the influence of the constituentsof fats and oils on cardiovascular disease, cancer and variousinflammatory conditions. High levels of cholesterol and saturated fattyacids in the diet are thought to increase the risk of heart disease andthis has led to nutritional advice to reduce the consumption ofcholesterol-rich saturated animal fats in favour of cholesterol-freeunsaturated plant oils (Liu et al., 2002a).

While dietary intake of cholesterol present in animal fats cansignificantly increase the levels of total cholesterol in the blood, ithas also been found that the fatty acids that comprise the fats and oilscan themselves have significant effects on blood serum cholesterollevels. Of particular interest is the effect of dietary fatty acids onthe undesirable low density lipoprotein (LDL) and desirable high densitylipoprotein (HDL) forms of cholesterol in the blood. In general,saturated fatty acids, particularly myristic acid (14:0) and palmiticacid (16:0), the principal saturates present in plant oils, have theundesirable property of raising serum LDL-cholesterol levels andconsequently increasing the risk of cardiovascular disease (Zock et al.,1994; Hu et al., 1997). However, it has become well established thatstearic acid (18:0), the other main saturate present in plant oils, doesnot raise LDL-cholesterol, and may actually lower total cholesterol(Bonanome and Grundy, 1988; Dougherty et al., 1995). Stearic acid istherefore generally considered to be at least neutral with respect torisk of cardiovascular disease (Tholstrup, et al., 1994). On the otherhand, unsaturated fatty acids, such as the monounsaturate oleic acid(18:1), have the beneficial property of lowering LDL-cholesterol(Mensink and Katan, 1989; Roche and Gibney, 2000), thus reducing therisk of cardiovascular disease.

Oil high in oleic acid also has many industrial uses such as, but notlimited to, lubricants often in the form of fatty acid esters, biofuels,raw materials for fatty alcohols, plasticizers, waxes, metal stearates,emulsifiers, personal care products, soaps and detergents, surfactants,pharmaceuticals, metal working additives, raw material for fabricsofteners, inks, transparent soaps, PVC stabilizer, alkyd resins, andintermediates for many other types of downstream oleochemicalderivatives.

Oil processors and food manufacturers have traditionally relied onhydrogenation to reduce the level of unsaturated fatty acids in oils,thereby increasing their oxidative stability in frying applications andalso providing solid fats for use in margarine and shortenings.Hydrogenation is a chemical process that reduces the degree ofunsaturation of oils by converting carbon-carbon double bonds intocarbon-carbon single bonds. Complete hydrogenation produces a fullysaturated fat. However, the process of partial hydrogenation results inincreased levels of both saturated fatty acids and monounsaturated fattyacids. Some of the monounsaturates formed during partial hydrogenationare in the trans isomer form (such as elaidic acid, a trans isomer ofoleic acid) rather than the naturally occurring cis isomer (Sebedio etal., 1994; Fernandez San Juan, 1995). In contrast to cis-unsaturatedfatty acids, trans-fatty acids are now known to be as potent as palmiticacid in raising serum LDL cholesterol levels (Mensink and Katan, 1990;Noakes and Clifton, 1998) and lowering serum HDL cholesterol (Zock andKatan, 1992), and thus contribute to increased risk of cardiovasculardisease (Ascherio and Willett, 1997). As a result of increased awarenessof the anti-nutritional effects of trans-fatty acids, there is now agrowing trend away from the use of hydrogenated oils in the foodindustry, in favour of fats and oils that are both nutritionallybeneficial and can provide the required functionality withouthydrogenation, in particular those that are rich in either oleic acidwhere liquid oils are required or stearic acid where a solid orsemi-solid fat is preferred.

There is a need for further lipids and oils with high oleic acid contentand sources thereof.

SUMMARY OF THE INVENTION

The present inventors have produced new lipid compositions and methodsof producing these lipids.

In a first aspect, the present invention provides lipid extracted froman oilseed, the lipid comprising triacylglycerols (TAG) which consist offatty acids esterified to glycerol, wherein

i) the fatty acids comprise palmitic acid and oleic acid,

ii) at least 95% by weight of the lipid is TAG,

iii) about 90% to about 95% by weight of the total fatty acid content ofthe lipid is oleic acid,

iv) less than about 3.1% by weight of the total fatty acid content ofthe lipid is palmitic acid, and

v) the lipid has an oleic desaturation proportion (ODP) of less thanabout 0.037 and/or a palmitic-linoleic-oleic value (PLO) of less thanabout 0.063.

In an embodiment, the lipid has one or more or all of the followingfeatures,

a) about 90% to about 94%, or about 91% to about 94%, or about 91% toabout 92%, or about 92%, or about 93%, by weight of the total fatty acidcontent of the lipid is oleic acid,

b) less than about 3%, or less than about 2.75%, or less than about2.5%, or about 3%, or about 2.75%, or about 2.5%, or about 2.3% byweight of the total fatty acid content of the lipid is palmitic acid,

c) about 0.1% to about 3%, or about 2% to about 3%, or about 3%, orabout 2%, by weight of the total fatty acid content of the lipid ispolyunsaturated fatty acids (PUFA),

d) less than about 3%, or less than about 2.5%, or less than about2.25%, or about 3%, or about 2.5%, or about 2.25%, by weight of thetotal fatty acid content of the lipid is linoleic acid,

e) less than about 1%, or less than about 0.5%, by weight of the totalfatty acid content of the lipid is α-linolenic acid (ALA), about 0.5% toabout 1% by weight of the total fatty acid content of the lipid is18:1Δ1,

g) the ODP of the fatty acid content of the lipid is about 0.033 toabout 0.01, or about 0.033 to about 0.016, or about 0.033 to about0.023, or is about 0.03, or about 0.02, or about 0.01,

h) the PLO value of the fatty acid content of the lipid is about 0.020to about 0.063, or about 0.020 to about 0.055, or about 0.020 to about0.050, or about 0.050 to about 0.055, or about 0.063, or about 0.055, orabout 0.050, or about 0.040, or about 0.030, or about 0.020,

i) about 90% to about 96%, or about 92% to about 96%, or about 93%, orabout 94%, by weight of the total fatty acid content of the lipid ismonounsaturated fatty acids,

j) the lipid has an oleic monounsaturation proportion (OMP) of less thanabout 0.02, or less than about 0.015, or about 0.005 to about 0.02,

k) the lipid is in the form of a purified oil, and

l) the lipid is non-hydrogenated.

In an embodiment,

1) about 91% to about 94% by weight of the total fatty acid content ofthe lipid is oleic acid,

2) less than about 2.75% by weight of the total fatty acid content ofthe lipid is palmitic acid,

3) less than about 3% by weight of the total fatty acid content of thelipid is linoleic acid,

4) α-linolenic acid is undetectable in the fatty acid content of thelipid,

5) the ODP of the fatty acid content of the lipid is about 0.033 toabout 0.023, or about 0.033 to about 0.018,

6) the PLO value of the fatty acid content of the lipid is about 0.020to about 0.063,

7) about 96%, or about 93%, or about 94%, by weight of the total fattyacid content of the lipid is monounsaturated fatty acids, and

8) the lipid has an oleic monounsaturation proportion (OMP) of less thanabout 0.02, or less than about 0.015, or about 0.005 to about 0.02.

In a further embodiment,

1) about 91% to about 94% by weight of the total fatty acid content ofthe lipid is oleic acid,

2) less than about 2.75% by weight of the total fatty acid content ofthe lipid is palmitic acid,

3) less than about 3% by weight of the total fatty acid content of thelipid is linoleic acid,

4) α-linolenic acid is undetectable in the fatty acid content of thelipid,

5) about 96%, or about 93%, or about 94%, by weight of the total fattyacid content of the lipid is monounsaturated fatty acids, and

6) the lipid has an oleic monounsaturation proportion (OMP) of less thanabout 0.02, or less than about 0.015, or about 0.005 to about 0.02.

In an embodiment, the PUFA is linoleic acid.

In an embodiment, α-linolenic acid is undetectable in the fatty acidcontent of the lipid.

In a further embodiment, about 55% to about 80%, or about 60% to about80%, or about 70% to about 80%, or at least about 60%, or at least about70%, or about 60%, or about 70%, or about 80%, of the TAG content of thelipid is triolein.

In another embodiment, less than about 5%, or less than about 2%, orabout 0.1% to about 5%, of the oleic acid content of the lipid is in theform of diacylglycerols (DAG).

In a further embodiment, the lipid is in the form of an oil, wherein atleast 90%, or least 95%, at least about 98%, or about 95% to about 98%,by weight of the oil is the lipid.

In a preferred embodiment, the oilseed is a non-photosynthetic oilseed.Examples of non-photosynthetic oilseed include, but are not necessarilylimited to, seed from safflower, sunflower, cotton or castor. In apreferred embodiment, the non-photosynthetic oilseed is safflower seed.

In an embodiment, the lipid further comprises one or more sterols.

In a further embodiment, the lipid is in the form of an oil, and whichcomprises less than about 5 mg of sterols/g of oil, or about 1.5 mg ofsterols/g of oil to about 5 mg of sterols/g of oil.

In an embodiment, the lipid comprises one or more or all of

a) about 1.5% to about 4.5%, or about 2.3% to about 4.5%, of the totalsterol content is ergost-7-en-3β-ol,

b) about 0.5% to about 3%, or about 1.5% to about 3%, of the totalsterol content is triterpenoid alcohol,

c) about 8.9% to about 20%, of the total sterol content isΔ7-stigmasterol/stigmast-7-en-3β-ol, and

d) about 1.7% to about 6.1% of the total sterol content isΔ7-avenasterol.

In a further embodiment, the lipid has a volume of at least 1 litreand/or a weight of at least 1 kg, and/or which was extracted fromoilseed obtained from field-grown plants.

In an embodiment, the lipid has been extracted from an oilseed bycrushing and comprises less than about 7% water by weight. In anotherembodiment, the lipid is purified lipid (solvent extracted and refined)and comprises less than about 0.1%, or less than about 0.05% water, byweight.

In another aspect, the present invention provides a compositioncomprising a first component which is lipid of the invention, and asecond component, preferably where the composition was produced bymixing the lipid with the second component.

As the skilled person will appreciate, the second component can beselected from a wide range of different compounds/compositions. In oneexample, the second component is a non-lipid substance such as anenzyme, a non-protein (non-enzymatic) catalyst or chemical (for example,sodium hydroxide, methanol or a metal), or one or more ingredients of afeed.

The present invention also provides a process for producing oil, theprocess comprising

i) obtaining an oilseed comprising, and/or which is capable of producinga plant which produces oilseed comprising, oil, wherein the oil contentof the oilseed is a lipid as defined in herein, and

ii) extracting oil from the oilseed so as to thereby produce the oil.

In a preferred embodiment, the oilseed comprises a first exogenouspolynucleotide which encodes a first silencing RNA which is capable ofreducing the expression of a Δ12 desaturase (FAD2) gene in a developingoilseed relative to a corresponding oilseed lacking the exogenouspolynucleotide, and wherein the polynucleotide is operably linked to apromoter which directs expression of the polynucleotide in thedeveloping oilseed.

In another aspect, the present invention provides a process forproducing oil, the process comprising

i) obtaining safflower seed whose oil content comprises, and/or which iscapable of producing a plant which produces seed whose oil contentcomprises, triacylglycerols (TAG) which consist of fatty acidsesterified to glycerol, and wherein

a) the fatty acids comprise palmitic acid and oleic acid,

b) at least 95% by weight of the oil content of the seed is TAG,

c) about 75% to about 95% by weight of the total fatty acids of the oilcontent of the seed is oleic acid,

d) less than about 5.1% by weight of the total fatty acids of the oilcontent of the seed is palmitic acid, and

e) the oil content of the seed has an oleic desaturation proportion(ODP) of less than about 0.17 and/or a palmitic-linoleic-oleic (PLO)value of less than about 0.26, and

ii) extracting oil from the safflower seed so as to thereby produce theoil,

wherein the safflower seed comprises a first exogenous polynucleotidewhich encodes a first silencing RNA which is capable of reducing theexpression of a Δ12 desaturase (FAD2) gene in a developing safflowerseed relative to a corresponding safflower seed lacking the exogenouspolynucleotide, and wherein the polynucleotide is operably linked to apromoter which directs expression of the polynucleotide in thedeveloping safflower seed.

In an embodiment, the oilseed or safflower seed comprises a secondexogenous polynucleotide which encodes a second silencing RNA which iscapable of reducing the expression of a palmitoyl-ACP thioesterase(FATB) gene in a developing oilseed or safflower seed relative to acorresponding oilseed or safflower seed lacking the second exogenouspolynucleotide, and wherein the second exogenous polynucleotide isoperably linked to a promoter which directs expression of thepolynucleotide in the developing oilseed or safflower seed.

In another embodiment, the oilseed or safflower seed comprises a thirdexogenous polynucleotide which encodes a third silencing RNA which iscapable of reducing the expression of a plastidial ω6 fatty aciddesaturase (FAD6) gene in a developing oilseed or safflower seedrelative to a corresponding oilseed or safflower seed lacking the thirdexogenous polynucleotide, and wherein the third exogenous polynucleotideis operably linked to a promoter which directs expression of thepolynucleotide in the developing oilseed or safflower seed.

In an embodiment,

1) the FAD2 gene is one or more or each of a CtFAD2-1 gene, a CtFAD2-2gene, and a CtFAD2-10 gene, preferably a CtFAD2-1 gene and/or a CtFAD2-2gene, and/or

2) the FATB gene is a CtFATB-3 gene, and/or

3) the FADE gene is a CtFAD6 gene.

In an embodiment, the oil content of the safflower seed has one or moreor all of the following features,

a) about 80% to about 94%, or about 85% to about 94%, or about 90% toabout 94%, or about 91% to about 94%, or about 91% to about 92%, orabout 92%, or about 93% by weight of the total fatty acids of the oilcontent of the seed is oleic acid,

b) less than about 5%, or less than about 4%, or less than about 3%, orless than about 2.75%, or less than about 2.5%, or about 3%, or about2.75%, or about 2.5% by weight of the total fatty acids of the oilcontent of the seed is palmitic acid,

c) about 0.1% to about 15%, or about 0.1% to about 10%, or about 0.1% toabout 7.5%, or about 0.1% to about 5%, or about 0.1% to about 3%, orabout 2% to about 3%, or about 3%, or about 2%, by weight of the totalfatty acids of the oil content of the seed is polyunsaturated fattyacids (PUFA),

d) less than about 15%, or less than about 10%, or less than about 5%,or less than about 3%, or less than about 2.5%, or less than about2.25%, or about 3%, or about 2.5%, or about 2.25%, by weight of thetotal fatty acids of the oil content of the seed is linoleic acid (LA),

e) about 80% to about 96%, or about 90% to about 96%, or about 92% toabout 96%, or about 93%, or about 94%, by weight of the total fatty acidcontent of the lipid is monounsaturated fatty acids,

f) the lipid has an oleic monounsaturation proportion (OMP) of less thanabout 0.05, or less than about 0.02, or less than about 0.015, or about0.005 to about 0.05, or about 0.005 to about 0.02,

g) the ODP of the oil content of the seed is about 0.17 to about 0.01,or about 0.15 to about 0.01, or about 0.1 to about 0.01, or about 0.075to about 0.01, or about 0.050 to about 0.01, or about 0.033 to about0.01, or about 0.033 to about 0.016, or about 0.033 to about 0.023, oris about 0.03, or about 0.02, or about 0.01, and

h) the PLO value of the oil content of the seed is about 0.20 to about0.026, or about 0.020 to about 0.2, or about 0.020 to about 0.15, orabout 0.020 to about 0.1, or about 0.020 and about 0.075, or about 0.050and about 0.055, or is about 0.05, or about 0.040, or about 0.030, orabout 0.020.

In an embodiment, the step of extracting the oil comprises crushing theoilseed or the safflower seed.

In yet another embodiment, the process further comprising a step ofpurifying the oil extracted from the oilseed or the safflower seed,wherein the purification step comprises one or more or all of the groupconsisting of: degumming, deodorising, decolourising, drying and/orfractionating the extracted oil, and/or removing at least some,preferably substantially all, waxes and/or wax esters from the extractedoil.

In another aspect, the present invention provides an oilseed whose oilcontent comprises, and/or which is capable of producing a plant whichproduces oilseed whose oil content comprises, triacylglycerols (TAG)which consist of fatty acids esterified to glycerol, and wherein

i) the fatty acids comprise palmitic acid and oleic acid,

ii) at least 95% by weight of the oil content of the oilseed is TAG,

iii) about 90% to about 95% by weight of the total fatty acids of theoil content of the oilseed is oleic acid,

iv) less than about 3.1% by weight of the total fatty acids of the oilcontent of the oilseed is palmitic acid, and

v) the oil content of the oilseed has an oleic desaturation proportion(ODP) of less than about 0.037 and/or a palmitic-linoleic-oleic (PLO)value of less than about 0.063.

In an embodiment, the oilseed is a non-photosynthetic oilseed,preferably seed from safflower, sunflower, cotton or castor.

In another embodiment, the oilseed comprises a first exogenouspolynucleotide which encodes a first silencing RNA which is capable orreducing the expression of a Δ12 desaturase (FAD2) gene in a developingoilseed relative to a corresponding oilseed lacking the exogenouspolynucleotide, and wherein the first exogenous polynucleotide isoperably linked to a promoter which directs expression of thepolynucleotide in the developing oilseed.

In yet a further aspect, the present invention provides a safflower seedwhose oil content comprises, and/or which is capable of producing aplant which produces seed whose oil content comprises, triacylglycerols(TAG) which consist of fatty acids esterified to glycerol, and wherein

i) the fatty acids comprise palmitic acid and oleic acid,

ii) at least 95% by weight of the oil content of the seed is TAG,

iii) about 75% to about 95% by weight of the total fatty acids of theoil content of the seed is oleic acid,

iv) less than about 5.1% by weight of the total fatty acids of the oilcontent of the seed is palmitic acid, and

v) the oil content of the seed has an oleic desaturation proportion(ODP) of less than about 0.17 and/or a palmitic-linoleic-oleic (PLO)value of less than about 0.26, and wherein the safflower seed comprisesa first exogenous polynucleotide which encodes a first silencing RNAwhich is capable of reducing the expression of a Δ12 desaturase (FAD2)gene in a developing safflower seed relative to a correspondingsafflower seed lacking the exogenous polynucleotide, and wherein thefirst exogenous polynucleotide is operably linked to a promoter whichdirects expression of the polynucleotide in the developing safflowerseed.

In an embodiment, the oil content of the safflower seed has one or moreof the following features,

a) about 80% to about 94%, or about 85% to about 94%, or about 90% toabout 94%, or about 91% to about 94%, or about 91% to about 92%, orabout 92%, or about 93% by weight of the total fatty acids of the oilcontent of the seed is oleic acid,

b) less than about 5%, or less than about 4%, or less than about 3%, orless than about 2.75%, or less than about 2.5%, or about 3%, or about2.75%, or about 2.5% by weight of the total fatty acids of the oilcontent of the seed is palmitic acid,

c) about 0.1% to about 15%, or about 0.1% to about 10%, or about 0.1% toabout 7.5%, or about 0.1% to about 5%, or about 0.1% to about 3%, orabout 2% to about 3%, or about 3%, or about 2% by weight of the totalfatty acids of the oil content of the seed is polyunsaturated fattyacids (PUFA),

d) less than about 15%, or less than about 10%, or less than about 5%,or less than about 3%, or less than about 2.5%, or less than about2.25%, or about 3%, or about 2.5%, or about 2.25%, by weight of thetotal fatty acids of the oil content of the seed is linoleic acid (LA),

e) about 80% to about 96%, or about 90% to about 96%, or about 92% toabout 96%, or about 93%, or about 94%, by weight of the total fatty acidcontent of the lipid is monounsaturated fatty acids,

f) the lipid has an oleic monounsaturation proportion (OMP) of less thanabout 0.05, or less than about 0.02, or less than about 0.015, or about0.005 to about 0.05, or about 0.005 to about 0.02,

g) the ODP of the oil content of the seed is about 0.17 to about 0.01,or about 0.15 to about 0.01, or about 0.1 to about 0.01, or about 0.075to about 0.01, or about 0.050 to about 0.01, or about 0.033 to about0.01, or about 0.033 to about 0.016, or about 0.033 to about 0.023, oris about 0.03, or about 0.02, or about 0.01, and

h) the PLO value of the oil content of the seed is about 0.020 to about0.26, or about 0.020 to about 0.2, or about 0.020 to about 0.15, orabout 0.020 to about 0.1, or about 0.020 and about 0.075, or about 0.050and about 0.055, or is about 0.050, or about 0.040, or about 0.030, orabout 0.020.

In a further embodiment, the oil content of the oilseed or safflowerseed is lipid which is further characterized by one or more of theabove-mentioned features.

In another embodiment, the oilseed or safflower seed comprises a secondexogenous polynucleotide which encodes a second silencing RNA which iscapable of reducing the expression of a palmitoyl-ACP thioesterase(FATB) gene in a developing oilseed or safflower seed relative to acorresponding oilseed or safflower seed lacking the second exogenouspolynucleotide, and wherein the second exogenous polynucleotide isoperably linked to a promoter which directs expression of thepolynucleotide in the developing oilseed or safflower seed.

In a further embodiment, the oilseed or safflower seed comprises a thirdexogenous polynucleotide which is capable of reducing the expression ofa plastidial ω6 fatty acid desaturase (FADE) gene in a developingoilseed or safflower seed relative to a corresponding oilseed orsafflower seed lacking the third exogenous polynucleotide, and whereinthe third exogenous polynucleotide is operably linked to a promoterwhich directs expression of the polynucleotide in the developing oilseedor safflower seed.

In another embodiment, the first silencing RNA reduces the expression ofmore than one endogenous gene encoding FAD2 in developing oilseed orsafflower seed and/or wherein the second silencing RNA reduces theexpression of more than one endogenous gene encoding FATB in developingoilseed or safflower seed.

In yet a further embodiment, the first exogenous polynucleotide andeither or both of the second exogenous polynucleotide and the thirdexogenous polynucleotide are covalently joined on a single DNA molecule,optionally with linking DNA sequences between the first, second and/orthe third exogenous polynucleotides.

In another embodiment, the first exogenous polynucleotide and either orboth of the second exogenous polynucleotide and the third exogenouspolynucleotide are under the control of a single promoter such that,when the first exogenous polynucleotide and the second exogenouspolynucleotide and/or the third exogenous polynucleotide are transcribedin the developing oilseed or safflower seed, the first silencing RNA andthe second silencing RNA and/or the third silencing RNA are covalentlylinked as parts of a single RNA transcript.

In another embodiment, the oilseed or safflower seed comprises a singletransfer DNA integrated into the genome of the oilseed or safflowerseed, and wherein the single transfer DNA comprises the first exogenouspolynucleotide and either or both of the second exogenous polynucleotideand the third exogenous polynucleotide.

Preferably, the oilseed or safflower seed is homozygous for the transferDNA.

In an embodiment, the first silencing RNA, the second silencing RNA andthe third silencing RNA are each independently selected from the groupconsisting of: an antisense polynucleotide, a sense polynucleotide, acatalytic polynucleotide, a microRNA and a double stranded RNA.

In a further embodiment, any one or more, preferably all, of thepromoters are seed specific, and preferably preferentially expressed inthe embryo of developing oilseed or safflower seed.

In another embodiment, the oilseed or safflower seed comprises one ormore mutations in one or more FAD2 genes, wherein the mutation(s) reducethe activity of the one or more FAD2 genes in developing oilseed orsafflower seed relative to a corresponding oilseed or safflower seedlacking the mutation(s).

In another embodiment, the oilseed or safflower seed comprises amutation of a FAD2 gene relative to a wild-type FAD2 gene in acorresponding oilseed or safflower seed, which mutation is a deletion,an insertion, an inversion, a frameshift, a premature translation stopcodon, or one or more non-conservative amino acid substitutions.

In a further embodiment, the mutation is a null mutation in the FAD2gene.

In another embodiment, at least one of the mutations is in a FAD2 genewhich encodes more FAD2 activity in the developing oilseed or safflowerseed lacking the mutation(s) than any other FAD2 gene in the developingoilseed or safflower seed.

In another embodiment, the seed is a safflower seed and the FAD2 gene isthe CtFAD2-1 gene. In this embodiment, it is also preferred that thefirst silencing RNA is at least capable or reducing the expression of aCtFAD2-2 gene.

In another embodiment, the seed is a safflower seed comprising an olallele of the CtFAD2-1 gene or an ol1 allele of the CtFAD2-1 gene, orboth alleles. In an embodiment, the ol allele or the ol1 allele of theCtFAD2-1 gene is present in the homozygous state.

In another embodiment, FAD2 protein is undetectable in the oilseed orsafflower seed.

In another embodiment, the seed is a safflower seed and the firstsilencing RNA reduces the expression of both CtFAD2-1 and CtFAD2-2genes.

In another embodiment, the seed is a safflower seed where

1) the FAD2 gene is one or more of a CtFAD2-1 gene, a CtFAD2-2 gene, anda CtFAD2-10 gene, preferably a CtFAD2-1 gene and/or a CtFAD2-2 gene,and/or

2) the FATB gene is a CtFATB-3 gene, and/or

3) the FADE gene is a CtFAD6 gene.

Also provided is an oilseed plant or safflower plant capable ofproducing the seed of the invention.

In an embodiment, the plant is transgenic and homozygous for eachexogenous polynucleotide, and/or comprises the first exogenouspolynucleotide and either or both of the second exogenous polynucleotideor the third exogenous polynucleotide.

In another aspect, the present invention provides a substantiallypurified and/or recombinant polypeptide which comprises amino acidshaving a sequence as provided in any one of SEQ ID NOs: 27 to 37, 44, 45or 48, a biologically active fragment thereof, or an amino acid sequencewhich is at least 40% identical to any one or more of SEQ ID NOs: 27 to37, 44, 45 or 48.

In an embodiment, the polypeptide which is a fatty acid modifyingenzyme, preferably an oleate Δ12 desaturase, Δ12-acetylenase, apalmitoleate Δ12 desaturase or a palmitoyl-ACP thioesterase (FATB).

In a further aspect, the present invention provides an isolated and/orexogenous polynucleotide comprising one or more of

i) nucleotides having a sequence as provided in any one of SEQ ID NOs: 1to 25, 40 to 43, 46 or 47,

ii) nucleotides having a sequence encoding a polypeptide of theinvention,

iii) nucleotides which hybridize to a sequence as provided in any one ofSEQ ID NOs: 1 to 25, 40 to 43, 46 or 47, and

iv) nucleotides having a sequence such that when expressed in a seed ofan oilseed plant reduces the expression of a gene encoding at least onepolypeptide of the invention.

In a particularly preferred embodiment, polynucleotide comprisesnucleotides having a sequence such that when expressed in a seed of anoilseed plant reduces the expression of a gene encoding at least onepolypeptide of the invention.

In an embodiment, the polynucleotide of part iv) comprises a sequence ofnucleotides provided in any one of SEQ ID NOs: 49 to 51 (where thymine(T) is uracil (U)).

In an embodiment, the polynucleotide of part iv) is selected from: anantisense polynucleotide, a sense polynucleotide, a catalyticpolynucleotide, a microRNA, a double stranded RNA (dsRNA) molecule or aprocessed RNA product thereof.

In a further embodiment, the polynucleotide is a dsRNA molecule, or aprocessed RNA product thereof, comprising at least 19 consecutivenucleotides which is at least 95% identical to the complement of any oneor more of SEQ ID NOs: 1 to 25, 40 to 43, 46, 47 or 49 to 51 (wherethymine (T) is uracil (U)).

In another embodiment, the dsRNA molecule is a microRNA (miRNA)precursor and/or wherein the processed RNA product thereof is a miRNA.

In yet a further embodiment, the polynucleotide is transcribed in adeveloping oilseed or safflower seed under the control of a singlepromoter, wherein the dsRNA molecule comprises complementary sense andantisense sequences which are capable of hybridising to each other,linked by a single stranded RNA region.

In yet a further embodiment, the polynucleotide, when present in adeveloping safflower seed,

i) reduces the expression of an endogenous gene encoding oleate Δ12desaturase (FAD2) in the developing seed, the FAD2 having an amino acidsequence as provided in any one or more of SEQ ID NOs: 27, 28 or 36,preferably at least one or both of SEQ ID NOs: 27 and 28;

ii) reduces the expression of an endogenous gene encoding palmitoyl-ACPthioesterase (FATB) in the developing seed, the FATB having an aminoacid sequence as provided in SEQ ID NO: 45; and/or

iii) reduces the expression of a gene encoding an ω6 fatty aciddesaturase (FAD6) in the developing seed, the FAD6 having an amino acidsequence as provided in SEQ ID NO: 48.

Also provided is a chimeric vector comprising the polynucleotide of theinvention, operably linked to a promoter.

In an embodiment, the promoter is functional in an oilseed, or is a seedspecific promoter, preferably is preferentially expressed in the embryoof a developing oilseed.

In another aspect, the present invention provides a recombinant cellcomprising an exogenous polynucleotide of the invention, and/or a vectorof the invention.

The cell can be any cell type such as, but not limited to, bacterialcell, yeast cell or plant cell.

Preferably, the cell is a plant cell, preferably a plant seed cell. Morepreferably, the plant cell is an oilseed plant cell. Even morepreferably, the plant cell is a non-photosynthetic seed cell, preferablya safflower, sunflower, cotton or castor seed cell.

In another aspect, the present invention provides a transgenic non-humanorganism comprising one or more of the polynucleotides of the invention,a vector of the invention, and a cell of the invention.

Preferably, the transgenic non-human organism of is a plant. Morepreferably, an oilseed plant.

In yet another aspect, the present invention provides a method ofproducing the cell of the invention, the method comprising the step ofintroducing the polynucleotide of the invention, and/or a vector of theinvention, into a cell.

Also provided is the use of the polynucleotide of the invention and/or avector of the invention to produce a recombinant cell.

In another aspect, the present invention provides a method of producinga transgenic oilseed plant which produces a seed of the invention, orseed thereof, the method comprising

i) introducing at least one polynucleotide of the invention and/or atleast one vector of the invention, into a cell of an oilseed plant,

ii) regenerating a transgenic plant from the cell, and

iii) optionally producing one or more progeny plants or seed thereoffrom the transgenic plant, thereby producing the transgenic oilseedplant or seed thereof.

In an embodiment, the seed is safflower seed of the invention.

In further embodiment, the one or more progeny plants or seed thereofcomprises

i) the first exogenous polynucleotide, and/or

ii) the second exogenous polynucleotide, and/or

iii) the third exogenous polynucleotide, preferably all three exogenouspolynucleotides.

In a further embodiment, the one or more progeny plants or seed thereofcomprises one or more mutations as defined above.

In yet a further aspect, the present invention provides a method ofobtaining an oilseed plant, the method comprising

i) crossing a first parental oilseed plant which comprises a firstpolynucleotide of the invention, or a first vector of the invention,with a second parental oilseed plant which comprises a secondpolynucleotide of the invention, or a first vector of the invention,

ii) screening progeny plants from the cross for the presence of bothpolynucleotides or both vectors; and

iii) selecting a progeny plant comprising both (a) the firstpolynucleotide or the first vector and (b) the second polynucleotide orthe second vector, and further having an increased proportion of oleicacid and a decreased proportion of palmitic acid in the oil content ofthe seed of the plant.

In a further aspect, the present invention provides a method ofgenotyping a safflower plant, the method comprising detecting a nucleicacid molecule of the plant, wherein the nucleic acid molecule is linkedto, and/or comprises at least part of, one or more of the CtFAD2-1,CtFAD2-2 or CtFAD2-10 genes, preferably at least one or both of theCtFAD2-1 and CtFAD2-2 genes, or at least the CtFAD2-1 gene, of asafflower plant.

In an embodiment, the method comprises determining the level ofexpression, and/or sequence, of one or more of the CtFAD2-1, CtFAD2-2 orCtFAD2-10 genes of the plant.

In an embodiment, the method comprises:

i) hybridising a second nucleic acid molecule to said nucleic acidmolecule of the plant,

ii) optionally hybridising at least one other nucleic acid molecule tosaid nucleic acid molecule of the plant; and

iii) detecting a product of said hybridising step(s) or the absence of aproduct from said hybridising step(s).

In yet a further embodiment, the second nucleic acid molecule is used asa primer to reverse transcribe or replicate at least a portion of thenucleic acid molecule of the plant.

The nucleic acid can detected using variety of well known techniquessuch as, but not limited to, restriction fragment length polymorphismanalysis, amplification fragment length polymorphism analysis,microsatellite amplification, nucleic acid sequencing, and/or nucleicacid amplification.

In an embodiment, the method detects the absence of presence of anallele of the CtFAD2-1 gene, preferably the ol allele.

In a further aspect, the present invention provides a method ofselecting a safflower plant from a population of safflower plants, themethod comprising;

i) genotyping said population of plants using a method of the invention,wherein said population of plants was obtained from a cross between twoplants of which at least one plant comprises an allele of a CtFAD2-1,CtFAD2-2 or CtFAD2-10 gene, preferably at least one or both of theCtFAD2-1 and CtFAD2-2 genes, or at least the CtFAD2-1 gene, whichconfers upon developing seed of said plant a reduced level of Δ12desaturase activity, relative to a corresponding seed of a safflowerplant lacking said allele, and

ii) selecting the safflower plant on the basis of the presence orabsence of said allele.

In a further aspect, the present invention provides a method ofintroducing an allele of a CtFAD2-1, CtFAD2-2 or CtFAD2-10 gene into asafflower plant lacking the allele, the method comprising;

i) crossing a first parent safflower plant with a second parentsafflower plant, wherein the second plant comprises said allele of aCtFAD2-1, CtFAD2-2 or CtFAD2-gene, and

ii) backcrossing the progeny of the cross of step i) with plants of thesame genotype as the first parent plant for a sufficient number of timesto produce a plant with a majority of the genotype of the first parentbut comprising said allele, wherein the allele confers upon developingseed of said plant a reduced level of Δ12 desaturase activity, relativeto a corresponding seed of a safflower plant lacking said allele, andwherein progeny plants are genotyped for the presence or absence of saidallele using a method of the invention.

Also provided is a transgenic plant, or progeny plants thereof, or seedthereof, produced using the method of the invention.

In a further aspect, the present invention provides a method ofproducing seed, the method comprising,

a) growing a plant of the invention, preferably in a field as part of apopulation of at least 1000 such plants, and

b) harvesting the seed.

In yet a further aspect, the present invention provides oil obtained orobtainable by one or more of the process of the invention, from theoilseed or safflower seed of the invention, from the plant or partthereof of the invention, from the cell of the invention, and/or fromthe non-human transgenic organism or part thereof of the invention.

In another aspect, the present invention provides a compositioncomprising one or more of the lipid of the invention, the oilseed orsafflower seed of the invention, the polypeptide of the invention, thepolynucleotide of the invention, the vector of the invention, the hostcell of the invention, or oil of the invention, and one or moreacceptable carriers.

Also provided is the use of one or more of the lipid of the invention,the composition of the invention, the process of the invention, theoilseed or safflower seed of the invention, the plant or part thereof ofthe invention, the host cell of the invention, the non-human transgenicorganism or part thereof of the invention, or oil of the invention, forthe manufacture of an industrial product.

In another aspect, the present invention provides a process forproducing an industrial product, the process comprising the steps of:

i) obtaining one or more of the lipid of the invention, the compositionof the invention, the oilseed or safflower seed of the invention, theplant or part thereof of the invention, the host cell of the invention,the non-human transgenic organism or part thereof of the invention, oroil of the invention,

ii) optionally physically processing the one or more of the lipid of theinvention, the composition of the invention, the oilseed or safflowerseed of the invention, the plant or part thereof of the invention, thehost cell of the invention, the non-human transgenic organism or partthereof of the invention, or oil of the invention, of step i), ii)converting at least some of the lipid of the invention, or lipid in oneor more of the composition of the invention, the oilseed or safflowerseed of the invention, the plant or part thereof of the invention, thehost cell of the invention, the non-human transgenic organism or partthereof of the invention, or oil of the invention, or the physicallyprocessed product of step ii), to the industrial product by applyingheat, chemical, or enzymatic means, or any combination thereof, to thelipid, and

iii) recovering the industrial product,

thereby producing the industrial product.

In yet a further aspect, the present invention provides a method ofproducing fuel, the method comprising

i) reacting one or more of the lipid of the invention, or lipid in oneor more of the invention, the oilseed or safflower seed of theinvention, the plant or part thereof of the invention, the host cell ofthe invention, the non-human transgenic organism or part thereof of theinvention, or oil of the invention, with an alcohol, optionally in thepresence of a catalyst, to produce alkyl esters, and

ii) optionally, blending the alkyl esters with petroleum based fuel.

In an embodiment, the alkyl esters are methyl esters.

In a further aspect, the present invention provides a method ofproducing a feedstuff, the method comprising admixing one or more of thelipid of the invention, the composition of the invention, oilseed orsafflower seed of the invention, the plant or part thereof of theinvention, the host cell according of the invention, the non-humantransgenic organism or part thereof of the invention, or oil of theinvention, with at least one other food ingredient.

Also provided is feedstuffs, cosmetics or chemicals comprising one ormore of the lipid of the invention, the composition of the invention,oilseed or safflower seed of the invention, the plant or part thereof ofthe invention, the host cell according of the invention, the non-humantransgenic organism or part thereof of the invention, or oil of theinvention,

In yet a further aspect, provided is a product produced from or usingone or more of the lipid of the invention, or lipid in one or more ofthe composition of the invention, the process of the invention, theoilseed or safflower seed of the invention, the plant or part thereof ofthe invention, the host cell of the invention, the non-human transgenicorganism or part thereof of the invention, or oil of the invention.

Any embodiment herein shall be taken to apply mutatis mutandis to anyother embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended for the purpose ofexemplification only. Functionally-equivalent products, compositions andmethods are clearly within the scope of the invention, as describedherein.

Throughout this specification, unless specifically stated otherwise orthe context requires otherwise, reference to a single step, compositionof matter, group of steps or group of compositions of matter shall betaken to encompass one and a plurality (i.e. one or more) of thosesteps, compositions of matter, groups of steps or group of compositionsof matter.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Phylogenetic comparison of amino acid sequences encoded bysafflower FAD2-like gene family and divergent FAD2-like enzymes fromother plants. The phylogenetic tree shown was generated by use of VectorNTI (invitrogen). Included in AAK26632.1; caACET, ABC00769.1; cpEPDX,CAA76156.1; haACET, ABC59684.1; dsACET, AA038036.1; dcACET, AA038033.1;hhACET, AA038031.1; haDES-2, AAL68982.1; haDES-3, AAL68983.1; luDES,ACF49507; haDES-1, AAL68982.1; ntDES, AAT72296.2; oeDES, AAW63040;siDES, AAF80560.1; ghDES-1, CAA65744.1; rcOH, AAC49010.1; atDES,AAM61113.1; pfOH:DES, AAC32755.1; plOH, ABQ01458.1; ghDES-4, AAQ16653.1;ghDES-2, CAA71199.1. (co, Calendula officinalis; ca, Crepis alpine; cp,Crepis palaestina; ha, Helianthus annuus; ds, Dimorphotheca sinuate; dc,Daucus carota; hh, Hedera helix; lu, Linum usitatissimum; nt, Nicotianatabacum; oe, Olea europaea; si, Sesamum indicum; rc, Ricinus communis;at, Arabidopsis thaliana; pf, Physaria fendleri; pl, Physarialindheimeri.

FIG. 2. Southern blot hybridisation analysis of CtFAD2-like genomicstructure in safflower genotype SU. Genomic DNA was digested with eightdifferent restriction enzymes prior to separation on an agarose gel.These enzymes were AccI (lane 1), BglII (2), BamHI (3), EcoRI (4), EcoRV(5), HindIII (6), XbaI (7) and XhoI (8). The blot was probed withradio-labelled entire coding region of CtFAD2-6 and washed at lowstringency conditions.

FIG. 3. GC-MS fatty acid analysis of fatty acid composition from yeastexpressing CtFAD2-1 (B), CtFAD2-2 (C), CtFAD2-9 (D), CtFAD2-10 (E) andCtFAD2-11 (F). Empty vector (A) and GC trace of the mixture of C18:2isomers (G).

FIG. 4. GC-MS fatty acid analysis of fatty acid composition afterCtFAD2-11 was transiently expressed in N. benthamiana leaves.

FIG. 5. RT-qPCR expression analysis of safflower CtFAD2 genes.

FIG. 6. Nucleotide comparison of a region of the CtFAD2-1 alleles fromwild-type SU (SEQ ID NO:56) and three high oleic genotypes, namelyS-317, CW99-OL and Lesaf496, showing a nucleotide deletion in the middleof the CtFAD2-1 coding region in the mutants (SEQ ID NO:57).

FIG. 7. DNA sequence comparison of the CtFAD2-1 5′UTR introns in thewild-type variety SU (SEQ ID NO:170) and the high oleic genotype S-317(SEQ ID NO:171). Boxed DNA sequences were used to design the perfect PCRmarkers for high oleic specific and wild type specific PCR products.

FIG. 8. Real time q-PCR analysis of CtFAD2-1 and CtFAD2-2 mRNA levels indeveloping embryos of three development stages, early (7 DPA), mid (15DPA) and late (20 DPA). The safflower varieties include wild-type SU,and three high oleic varieties: S-317, CW99-OL and Lesaf496.

FIG. 9. Real time qPCR analysis of CtFatB genes in leaf, root, anddeveloping embryos of safflower variety SU. Em-1 (early stage), Em-2(middle stage), and Em-3 (late stage).

FIG. 10. A dendrogram showing the phylogenetic relationship between thesafflower FAD6 sequences and representative FAD6 plastidial Δ12desaturase identified in higher plants. Jatropha curcas (EU106889); Oleaeuropaea (AY733075); Populus trichocarpa (EF147523); Arabidopsisthaliana (AY079039); Descuriana sophia (EF524189); Glycine max(AK243928); Brassica napus (L29214); Portulaca oleracea (EU376530);Arachis hypogaea (FJ768730); Ginkgo biloba (HQ694563).

FIG. 11. Diacylglycerol (DAG) composition (mol %) in S317 versusS317+603.9 by LC-MS analysis of single seed.

FIG. 12. Triacylglycerol (TAG) composition (mol %) in S317 versusS317+603.9 by LC-MS analysis of single seed.

FIG. 13. Oleic acid content of safflower varieties under fieldconditions at Narrabri in the Australian summer of 2011/2012.

FIG. 14. (A) Basic phytosterol structure with ring and side chainnumbering. (B) Chemical structures of some of the phytosterols.

KEY TO THE SEQUENCE LISTING

SEQ ID NO: 1—cDNA encoding safflower FAD2-1.

SEQ ID NO: 2—cDNA encoding safflower FAD2-2.

SEQ ID NO: 3—cDNA encoding safflower FAD2-3.

SEQ ID NO: 4—cDNA encoding safflower FAD2-4.

SEQ ID NO: 5—cDNA encoding safflower FAD2-5.

SEQ ID NO: 6—cDNA encoding safflower FAD2-6.

SEQ ID NO: 7—cDNA encoding safflower FAD2-7.

SEQ ID NO: 8—cDNA encoding safflower FAD2-8.

SEQ ID NO: 9—cDNA encoding safflower FAD2-9.

SEQ ID NO: 10—cDNA encoding safflower FAD2-10.

SEQ ID NO: 11—cDNA encoding safflower FAD2-11.

SEQ ID NO: 12—Open reading frame encoding safflower FAD2-1.

SEQ ID NO: 13—Open reading frame encoding safflower FAD2-2.

SEQ ID NO: 14—Open reading frame encoding safflower FAD2-3.

SEQ ID NO: 15—Open reading frame encoding safflower FAD2-4.

SEQ ID NO: 16—Open reading frame encoding safflower FAD2-5.

SEQ ID NO: 17—Open reading frame encoding safflower FAD2-6.

SEQ ID NO: 18—Open reading frame encoding safflower FAD2-7.

SEQ ID NO: 19—Open reading frame encoding safflower FAD2-8.

SEQ ID NO: 20—Open reading frame encoding safflower FAD2-9.

SEQ ID NO: 21—Open reading frame encoding safflower FAD2-10.

SEQ ID NO: 22—Open reading frame encoding safflower FAD2-11.

SEQ ID NO: 23—Intron sequence of safflower FAD2-1 gene.

SEQ ID NO: 24—Intron sequence of safflower FAD2-2 gene.

SEQ ID NO: 25—Intron sequence of safflower FAD2-10 gene.

SEQ ID NO: 26—cDNA encoding truncated safflower FAD2-1 (HO mutant).

SEQ ID NO: 27—Safflower FAD2-1.

SEQ ID NO: 28—Safflower FAD2-2.

SEQ ID NO: 29—Safflower FAD2-3.

SEQ ID NO: 30—Safflower FAD2-4.

SEQ ID NO: 31—Safflower FAD2-5.

SEQ ID NO: 32—Safflower FAD2-6.

SEQ ID NO: 33—Safflower FAD2-7.

SEQ ID NO: 34—Safflower FAD2-8.

SEQ ID NO: 35—Safflower FAD2-9.

SEQ ID NO: 36—Safflower FAD2-10.

SEQ ID NO: 37—Safflower FAD2-11.

SEQ ID NO:38—Truncated safflower FAD2-1 (HO mutant).

SEQ ID NO: 39—cDNA of safflower FATB-1.

SEQ ID NO: 40—cDNA encoding safflower FATB-2.

SEQ ID NO: 41—cDNA encoding safflower FATB-3.

SEQ ID NO: 42—Open reading frame encoding safflower FATB-2.

SEQ ID NO: 43—Open reading frame encoding safflower FATB-3.

SEQ ID NO: 44—Safflower FATB-2.

SEQ ID NO: 45—Safflower FATB-3.

SEQ ID NO: 46—cDNA encoding safflower FAD6.

SEQ ID NO: 47—Open reading frame encoding safflower FAD6.

SEQ ID NO: 48—Safflower FAD6.

SEQ ID NO: 49—CtFAD2-2 sequence used in RNA silencing construct.

SEQ ID NO: 50—CtFATB-3 sequence used in RNA silencing construct.

SEQ ID NO: 51—CtFAD6 sequence used in RNA silencing construct.

SEQ ID NO: 52—Arabidopsis thaliana oleosin promoter.

SEQ ID NO: 53—Flax linin promoter.

SEQ ID NO: 54—Nos polyadenylation signal.

SEQ ID NO: 55—Ocs polyadenylation signal.

SEQ ID NO: 56—Wild type CtFAD2-1 sequence corresponding to region of olallele.

SEQ ID NO: 57—Ol allele CtFAD2-1 sequence with frameshift (same forS-317, CW99-OL and LeSaf496).

SEQ ID NO's 58 to 158 —Oligonucleotide primers.

SEQ ID NO's 159 to 169—Motifs of CtFAD2 enzymes.

SEQ ID NO: 170—Wild type safflower variety SU CtFAD2-1 5′UTR intron.

SEQ ID NO: 171—High oleic acid safflower variety S-317 CtFAD2-1 5′UTRintron.

DETAILED DESCRIPTION OF THE INVENTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in cell culture,molecular genetics, immunology, immunohistochemistry, protein chemistry,and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present invention are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present).

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either“X and Y” or “X or Y” and shall be taken to provide explicit support forboth meanings or for either meaning.

As used herein, the term about, unless stated to the contrary, refers to+/−10%, more preferably +/−5%, more preferably +/−4%, more preferably+/−3%, more preferably +/−2%, more preferably +/−1.5%, more preferably+/−1%, even more preferably +/−0.5%, of the designated value.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

As used herein, the term “extracted lipid” refers to a lipid compositionwhich comprises at least 60% (w/w) lipid and which has been extracted,for example by crushing, from a transgenic organism or part thereof.Furthermore, as used herein, the term “extracted oil” refers to an oilcomposition which comprises at least 60% (w/w) oil and which has beenextracted from a transgenic organism or part thereof.

As used herein, the term “purified” when used in connection with lipidor oil of the invention typically means that that the extracted lipid oroil has been subjected to one or more processing steps of increase thepurity of the lipid/oil component. For example, a purification step maycomprise one or more or all of the group consisting of: degumming,deodorising, decolourising, drying and/or fractionating the extractedoil. However, as used herein, the term “purified” does not include atransesterification process or other process which alters the fatty acidcomposition of the lipid or oil of the invention so as to increase theoleic acid content as a percentage of the total fatty acid content.Expressed in other words, the fatty acid composition of the purifiedlipid or oil is essentially the same as that of the unpurified lipid oroil. The fatty acid composition of the extracted lipid or oil, such asfor example the oleic, linoleic and palmitic acid contents, isessentially the same as the fatty acid composition of the lipid or oilin the plant seed from which it is obtained. In this context,“essentially the same” means+/−1%, or, preferably, +/−0.5%.

As used herein, the term “oleic desaturation proportion” or “ODP” refersto a calculation which involves dividing the relative amount of linoleicacid and α-linolenic acid expressed as a percentage of the lipid fattyacid composition by the sum of the relative amounts of oleic acid,linoleic and α-linolenic acids, each expressed as percentages. Theformula is:ODP=(% linoleic+%α-linolenic)/(% oleic+% linoleic+%α-linolenic)For example, TG603.12(5) of Example 15 has a total linoleic acid andα-linolenic acid content of 2.15% and an linoleic acid, α-linolenic acidoleic acid content of 93.88% making the ODP 0.0229.

As used herein, the term “palmitic-linoleic-oleic value” or “PLO” refersto a calculation which involves dividing the relative amount of linoleicacid and palmitic acid expressed as a percentage of the lipid fatty acidcomposition by the relative amount of oleic acid expressed as apercentage. The formula is:PLO=(% palmitic+% linoleic)/% oleicFor example, TG603.12(5) of Example 15 has a total linoleic acid andpalmitic content of 4.71% and an oleic acid content of 91.73% making thePLO 0.0513.

As used herein, the term “oleic monounsaturation proportion” or “OMP”refers to a calculation which involves dividing the relative amount ofnon-oleic monounsaturated fatty acids expressed as a percentage of thelipid fatty acid composition by the relative amount of oleic acidexpressed as a percentage. The formula is:OMP=(% monounsaturated fatty acids−% oleic)/% oleicFor example, TG603.12(5) of Example 15 has a total monounsaturated fattyacid content excluding oleic acid (0.84% C18:1Δ11+0.29% C20:1) of 1.13%and an oleic acid content of 91.73% making the OMP 0.0123.

The term “corresponding” refers to a cell, or plant or part thereof(such as a seed) that has the same or similar genetic background as acell, or plant or part thereof (seed) of the invention but that has notbeen modified as described herein (for example, the cell, or plant orpart thereof lacks an exogenous polynucleotide of the invention). Acorresponding cell or, plant or part thereof (seed) can be used as acontrol to compare, for example, one or more of the amount of oleic acidproduced, FAD2 activity, FATB activity or FAD6 activity with a cell, orplant or part thereof (seed) modified as described herein. A personskilled in the art is able to readily determine an appropriate“corresponding” cell, plant or part thereof (seed) for such acomparison.

As used herein, the term “seedoil” refers to a composition obtained fromthe seed of a plant which comprises at least 60% (w/w) lipid, orobtainable from the seed if the seedoil is still present in the seed.That is, seedoil of, or obtained using, the invention includes seedoilwhich is present in the seed or portion thereof such as cotyledons orembryo, unless it is referred to as “extracted seedoil” or similar termsin which case it is oil which has been extracted from the seed. Theseedoil is preferably extracted seedoil. Seedoil is typically a liquidat room temperature. Preferably, the total fatty acid (TFA) content inthe seedoil is >70% C18 fatty acids, preferably >90% oleic acid(C18:1Δ9). The fatty acids are typically in an esterified form such asfor example, TAG, DAG, acyl-CoA or phospholipid. Unless otherwisestated, the fatty acids may be free fatty acids and/or in an esterifiedform. In an embodiment, at least 50%, more preferably at least 70%, morepreferably at least 80%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, more preferably at least93%, more preferably at least 94%, more preferably at least 95%, morepreferably at least 96%, more preferably at least 97%, more preferablyat least 98%, more preferably at least 99% of the fatty acids in seedoilof the invention can be found as TAG. In an embodiment, seedoil of theinvention is “substantially purified” or “purified” oil that has beenseparated from one or more other lipids, nucleic acids, polypeptides, orother contaminating molecules with which it is associated in the seed orin a crude extract. It is preferred that the substantially purifiedseedoil is at least 60% free, more preferably at least 75% free, andmore preferably, at least 90% free from other components with which itis associated in the seed or extract. Seedoil of the invention mayfurther comprise non-fatty acid molecules such as, but not limited to,sterols (see Example 17). In an embodiment, the seedoil is safflower oil(Carthamus tinctorius), sunflower oil (Helianthus annus), cottonseed oil(Gossypium hirsutum), castor oil (Ricinus communis), canola oil(Brassica napus, Brassica rapa ssp.), mustard oil (Brassica juncea),other Brassica oil (e.g., Brassica napobrassica, Brassica camelina),linseed oil (Linum usitatissimum), soybean oil (Glycine max), corn oil(Zea mays), tobacco oil (Nicotiana tabacum), peanut oil (Arachishypogaea), palm oil (Elaeis guineensis), coconut oil (Cocos nucifera),avocado oil (Persea americana), olive oil (Olea europaea), cashew oil(Anacardium occidentale), macadamia oil (Macadamia intergrifolia),almond oil (Prunus amygdalus), oat seed oil (Avena sativa), rice oil(Oryza sativa or Oryza glaberrima), camelina oil (Camelina sativa),crambe oil (Crambe abyssinica) or Arabidopsis seed oil (Arabidopsisthaliana). Seedoil may be extracted from seed by any method known in theart. This typically involves extraction with nonpolar solvents such ashexane, diethyl ether, petroleum ether, chloroform/methanol or butanolmixtures, generally associated with first crushing or rolling of theseeds. Lipids associated with the starch in the grain may be extractedwith water-saturated butanol. The seedoil may be “de-gummed” by methodsknown in the art to remove polysaccharides and/or phospholipids ortreated in other ways to remove contaminants or improve purity,stability, or colour. The TAGs and other esters in the seedoil may behydrolysed to release free fatty acids such as by acid or alkalitreatment or by the action of lipases, or the seedoil hydrogenated,treated chemically, or enzymatically as known in the art. However, oncethe seedoil is processed so that it no longer comprises the TAG, it isno longer considered seedoil as referred to herein.

The free and esterified sterol (for example, sitosterol, campesterol,stigmasterol, brassicasterol, Δ5-avenasterol, sitostanol, campestanol,and cholesterol) concentrations in the purified and/or extracted lipidor oil may be as described in Phillips et al. (2002) and/or as providedin Example 17. Sterols in plant oils are present as free alcohols,esters with fatty acids (esterified sterols), glycosides and acylatedglycosides of sterols. Sterol concentrations in naturally occurringvegetable oils (seedoils) ranges up to a maximum of about 1100 mg/100 g.Hydrogenated palm oil has one of the lowest concentrations of naturallyoccurring vegetable oils at about 60 mg/100 g. The recovered orextracted seedoils of the invention preferably have between about 100and about 1000 mg total sterol/100 g of oil. For use as food or feed, itis preferred that sterols are present primarily as free or esterifiedforms rather than glycosylated forms. In the seedoils of the presentinvention, preferably at least 50% of the sterols in the oils arepresent as esterified sterols, except for soybean seedoil which hasabout 25% of the sterols esterified. The safflower seedoil of theinvention preferably has between about 150 and about 400 mg totalsterol/100 g, typically about 300 mg total sterol/100 g of seedoil, withsitosterol the main sterol. The canola seedoil and rapeseed oil of theinvention preferably have between about 500 and about 800 mg totalsterol/100 g, with sitosterol the main sterol and campesterol the nextmost abundant. The corn seedoil of the invention preferably has betweenabout 600 and about 800 mg total sterol/100 g, with sitosterol the mainsterol. The soybean seedoil of the invention preferably has betweenabout 150 and about 350 mg total sterol/100 g, with sitosterol the mainsterol and stigmasterol the next most abundant, and with more freesterol than esterified sterol. The cottonseed oil of the inventionpreferably has between about 200 and about 350 mg total sterol/100 g,with sitosterol the main sterol. The coconut oil and palm oil of theinvention preferably have between about 50 and about 100 mg totalsterol/100 g, with sitosterol the main sterol. The peanut seedoil of theinvention preferably has between about 100 and about 200 mg totalsterol/100 g, with sitosterol the main sterol. The sesame seedoil of theinvention preferably has between about 400 and about 600 mg totalsterol/100 g, with sitosterol the main sterol. The sunflower seedoil ofthe invention preferably has between about 200 and 400 mg totalsterol/100 g, with sitosterol the main sterol.

As used herein, the term “fatty acid” refers to a carboxylic acid with along aliphatic tail of at least 8 carbon atoms in length, eithersaturated or unsaturated. Typically, fatty acids have a carbon-carbonbonded chain of at least 12 carbons in length. Most naturally occurringfatty acids have an even number of carbon atoms because theirbiosynthesis involves acetate which has two carbon atoms. The fattyacids may be in a free state (non-esterified) or in an esterified formsuch as part of a TAG, DAG, MAG, acyl-CoA (thio-ester) bound, or othercovalently bound form. When covalently bound in an esterified form, thefatty acid is referred to herein as an “acyl” group. The fatty acid maybe esterified as a phospholipid such as a phosphatidylcholine (PC),phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol,phosphatidylinositol, or diphosphatidylglycerol. Saturated fatty acidsdo not contain any double bonds or other functional groups along thechain. The term “saturated” refers to hydrogen, in that all carbons(apart from the carboxylic acid [—COOH] group) contain as many hydrogensas possible. In other words, the omega (ω) end contains 3 hydrogens(CH3-) and each carbon within the chain contains 2 hydrogens (—CH2-).Unsaturated fatty acids are of similar form to saturated fatty acids,except that one or more alkene functional groups exist along the chain,with each alkene substituting a singly-bonded “—CH2-CH2-” part of thechain with a doubly-bonded “—CH═CH—” portion (that is, a carbon doublebonded to another carbon). The two next carbon atoms in the chain thatare bound to either side of the double bond can occur in a cis or transconfiguration.

As used herein, the terms “polyunsaturated fatty acid” or “PUFA” referto a fatty acid which comprises at least 12 carbon atoms in its carbonchain and at least two alkene groups (carbon-carbon double bonds).

As used herein, the term “monounsaturated fatty acids” refers to fattyacids that have a single double bond in their acyl chain, such as oleicacid (C18:1Δ9), C18:1D11 and C20:1.

“Triacylglyceride” or “TAG” is glyceride in which the glycerol isesterified with three fatty acids. In the Kennedy pathway of TAGsynthesis, DAG is formed as described above, and then a third acyl groupis esterified to the glycerol backbone by the activity of DGAT.Alternative pathways for formation of TAG include one catalysed by theenzyme PDAT and the MGAT pathway (PCT/AU2011/000794).

“Diacylglyceride” or “DAG” is glyceride in which the glycerol isesterified with two fatty acids. As used herein, DAG comprises ahydroxyl group at a sn-1,3 or sn-1,2/2,3 position, and therefore DAGdoes not include phosphorylated molecules such as PA or PC. DAG is thusa component of neutral lipids in a cell. In the Kennedy pathway of DAGsynthesis, the precursor sn-glycerol-3-phosphate (G-3-P) is esterifiedto two acyl groups, each coming from a fatty acid coenzyme A ester, in afirst reaction catalysed by a glycerol-3-phosphate acyltransferase(GPAT) at position sn-1 to form LysoPA, followed by a second acylationat position sn-2 catalysed by a lysophosphatidic acid acyltransferase(LPAAT) to form phosphatidic acid (PA). This intermediate is thende-phosphorylated to form DAG. In an alternative anabolic pathway, DAGmay be formed by the acylation of either sn-1 MAG or preferably sn-2MAG, catalysed by MGAT. DAG may also be formed from TAG by removal of anacyl group by a lipase, or from PC essentially by removal of a cholineheadgroup by any of the enzymes CPT, PDCT or PLC.

As used herein, the term “desaturase” refers to an enzyme which iscapable of introducing a carbon-carbon double bond into the acyl groupof a fatty acid substrate which is typically in an esterified form suchas, for example, fatty acid CoA esters. The acyl group may be esterifiedto a phospholipid such as phosphatidylcholine (PC), or to acyl carrierprotein (ACP), to CoA, or in a preferred embodiment to PC. Desaturasesgenerally may be categorized into three groups accordingly. In oneembodiment, the desaturase is a front-end desaturase.

As used herein, the terms “Δ12 desaturase” and “FAD2” refer to amembrane bound Δ12 fatty acid desturase which performs a desaturasereaction converting oleic acid (18:1^(Δ9)) to linoleic acid(C18:2^(Δ9,12)). Thus, the term “Δ12 desaturase activity” refers to theconversion of oleic acid to linoleic acid. These fatty acids may be inan esterified form, such as, for example, as part of a phospholipid,preferably in the form of PC. In an embodiment, a FAD2 enzyme as definedherein comprises three histidine-rich motifs (His boxes) (see Table 5for examples of His boxes of enzymes of the invention). Such His-richmotifs are highly conserved in FAD2 enzymes and have been implicated inthe formation of the diiron-oxygen complex used in biochemical catalysis(Shanklin et al., 1998).

As used herein, the terms “FAD2-1” and “CtFAD2-1” and variations thereofrefer to a safflower FAD2 polypeptide whose amino acid sequence isprovided as SEQ ID NO:27, such as a polypeptide encoded by nucleotideshaving a sequence provided as SEQ ID NO:12. As used herein, a FAD2-1gene is a gene encoding such a polypeptide or a mutant allele thereof.These terms also include naturally occurring or artificially induced orproduced variants of the sequences provided. In an embodiment, FAD2-1 ofthe invention comprises an amino acid sequence which is at least 95%identical, more preferably at least 99% identical, to the sequenceprovided as SEQ ID NO:27. CtFAD2-1 genes include alleles which aremutant, that is, that encode polypeptides with altered desaturaseactivity such as reduced activity, or do not encode functionalpolypeptides (null alleles). Such alleles may be naturally occurring orinduced by artificial mutagenesis. An example of such an allele is theFAD2-1 ol allele described herein.

As used herein, the terms “FAD2-2” and “CtFAD2-2” and variations thereofrefer to a safflower FAD2 polypeptide whose amino acid sequence isprovided as SEQ ID NO:28, such as a polypeptide encoded by nucleotideshaving a sequence provided as SEQ ID NO:13. As used herein, a FAD2-2gene is a gene encoding such a polypeptide or a mutant allele thereof.These terms also include naturally occurring or artificially induced orproduced variants of the sequences provided. In an embodiment, FAD2-2 ofthe invention comprises an amino acid sequence which is at least 95%identical, more preferably at least 99% identical, to the sequenceprovided as SEQ ID NO:28. CtFAD2-2 genes include alleles which aremutant, that is, that encode polypeptides with altered desaturaseactivity such as reduced activity, or do not encode functionalpolypeptides (null alleles). Such alleles may be naturally occurring orinduced by artificial mutagenesis.

As used herein, the terms “FAD2-10” and “CtFAD2-10” and variationsthereof refer to a safflower FAD2 polypeptide whose amino acid sequenceis provided as SEQ ID NO:36, such as a polypeptide encoded bynucleotides having a sequence provided as SEQ ID NO:21. As used herein,a FAD2-10 gene is a gene encoding such a polypeptide or a mutant allelethereof. These terms also include naturally occurring or artificiallyinduced or produced variants of the sequences provided. In anembodiment, FAD2-10 of the invention comprises an amino acid sequencewhich is at least 95% identical, more preferably at least 99% identical,to the sequence provided as SEQ ID NO:36. CtFAD2-10 genes includealleles which are mutant, that is, that encode polypeptides with altereddesaturase activity such as reduced activity, or do not encodefunctional polypeptides (null alleles). Such alleles may be naturallyoccurring or induced by artificial mutagenesis.

As used herein, the term “palmitoyl-ACP thioesterase” and “FATB” referto a protein which hydrolyses palmitoyl-ACP to produce free palmiticacid. Thus, the term “palmitoyl-ACP thioesterase activity” refers to thehydrolysis of palmitoyl-ACP to produce free palmitic acid.

As used herein, the terms “FATB-3” and “CtFATB-3” and variations thereofrefer to a safflower FATB polypeptide whose amino acid sequence isprovided as SEQ ID NO:45, such as a polypeptide encoded by nucleotideshaving a sequence provided as SEQ ID NO:43. As used herein, a FATB-3gene is a gene encoding such a polypeptide or a mutant allele thereof.These terms also include naturally occurring or artificially induced orproduced variants of the sequences provided. In an embodiment, FATB-3 ofthe invention comprises an amino acid sequence which is at least 95%identical, more preferably at least 99% identical, to the sequenceprovided as SEQ ID NO:45. CtFATB-3 genes include alleles which aremutant, that is, that encode polypeptides with altered palmitoyl-ACPthioesterase activity such as reduced activity, or do not encodefunctional polypeptides (null alleles). Such alleles may be naturallyoccurring or induced by artificial mutagenesis.

As used herein, “plastidial ω6 fatty acid desaturase”, variationsthereof, and “FAD6” refer to a chloroplast enzyme that desaturates 16:1and 18:1 fatty acids to 16:2 and 18:2, respectively, on all 16:1- or18:1-containing chloroplast membrane lipids including phosphatidylglycerol, monogalactosyldiacylglycerol, digalactosyl-diacylglycerol, andsulfoguinovosyldiacylglycerol.

As used herein, the terms “FAD6” and “CtFAD6” and variations thereofrefer to a safflower FAD6 polypeptide whose amino acid sequence isprovided as SEQ ID NO:48, such as a polypeptide encoded by nucleotideshaving a sequence provided as SEQ ID NO:47. As used herein, a FAD6 geneis a gene encoding such a polypeptide or a mutant allele thereof. Theseterms also include naturally occurring or artificially induced orproduced variants of the sequences provided. In an embodiment, FAD6 ofthe invention comprises an amino acid sequence which is at least 95%identical, more preferably at least 99% identical, to the sequenceprovided as SEQ ID NO:48. CtFAD6 genes include alleles which are mutant,that is, that encode polypeptides with altered desaturase activity suchas reduced activity, or do not encode functional polypeptides (nullalleles). Such alleles may be naturally occurring or induced byartificial mutagenesis.

As used herein, the term “acetylenase” or “fatty acid acetylenase”refers to an enzyme that introduces a triple bond into a fatty acidresulting in the production of an acetylenic fatty acid.

The term “Ct” is used herein before terms such as FAD2, FATB and FAD6 toindicate the enzyme/gene is from safflower.

As used herein, the term “silencing RNA which is capable of reducing theexpression of” and variants thereof, refers to a polynucleotide thatencodes an RNA molecule that reduces (down-regulates) the productionand/or activity (for example, encoding an siRNA, hpRNAi), or itself downregulates the production and/or activity (for example, is an siRNA whichcan be delivered directly to, for example, a cell) of an endogenousenzyme for example, a Δ12 desaturase, a palmitoyl-ACP thioesterase, aplastidial ω6 fatty acid desaturase, or a combination of two or more orall three thereof. In an embodiment, the silencing RNA is an exogenousRNA which is produced from a transgene in the cell and whichtranscriptionally and/or post-transcriptionally reduces the amount ofthe endogenous enzyme that is produced in the cell, such as by reducingthe amount of mRNA encoding the endogenous enzyme or reducing itstranslation. The silencing RNA is typically an RNA of 21-24 nucleotidesin length which is complementary to the endogenous mRNA and which may beassociated with a silencing complex known as a RISC in the cell.

As used herein, the term “mutation(s) reduce the activity of” refers tonaturally occurring or man-made mutants (such as produced by chemicalmutagenesis or site-specific mutagenesis) which have lower levels of thedefined enzymatic activity (for example, FAD2 enzymatic activity in theseed) when compared to phenotypically normal seeds (for example, seedswhich produce FAD2 enzymes which comprise the amino acid sequencesprovided as SEQ ID NOs 27, 28 and 36). Examples of phenotypically normalsafflower varieties include, but are not limited to, Centennial, Finch,Nutrasaff and Cardinal. The first identified high oleic trait insafflower, found in a safflower introduction from India, was controlledby a partially recessive allele designated ol at a single locus OL(Knowles and Hill, 1964). As described herein, the OL locus correspondsto the CtFAD2-1 gene. The oleic acid content of seedoil in ololgenotypes was usually 71-75% for greenhouse-grown plants (Knowles,1989). Knowles (1968) incorporated the ol allele into a safflowerbreeding program and released the first high oleic (HO) safflowervariety “UC-1” in 1966 in the US, which was followed by the release ofimproved varieties “Oleic Leed” and the Saffola series including Saffola317 (S-317), S-517 and S-518. The high oleic (olol) genotypes wererelatively stable in the oleic acid level when grown at differenttemperatures in the field (Bartholomew, 1971). In addition, Knowles(1972) also described a different allele ol₁ at the same locus, whichproduced in homozygous condition between 35 and 50% oleic acid. Incontrast to olol genotype, the ol₁ol₁ genotype showed a strong responseto temperature (Knowles, 1972). As determined herein, the allele of theol mutation which confers reduced FAD2-1 activity (and overall FAD2activity) in safflower seed is a mutant FAD2-1 gene comprising theframeshift mutation (due to deletion of a single nucleotide) depicted inFIG. 6 (see also Example 7 and SEQ ID NOs 26 and 38).

As used herein, the phrase “which is capable of producing a plant whichproduces seed whose oil content comprises” or “which is capable ofproducing a plant which produces oilseed whose oil content” orvariations thereof means that the plant produced from the seed,preferably an oilseed plant and more preferably a safflower plant, hasthe capacity to produce the oil with the defined components when grownunder optimal conditions, for instance in greenhouse conditions such asthose referred to in the Examples. When in possession of seed from aplant, it is routine to grow a progeny plant from at least one of theseeds under suitable greenhouse conditions and test the oil content andfatty acid composition in seedoil from the progeny plant using standardprocedures such as those described herein. Accordingly, as the skilledperson would understand whilst seed grown in a field may not meet all ofthe requirements defined herein due to unfavourable conditions in aparticular year such heat, cold, drought, flooding, frost, pest stressesetc, such seed are nonetheless encompassed by the present inventionbecause the seed is capable of producing a progeny plant which producesthe defined oil content or fatty acid composition when grown under morefavourable conditions.

As used herein, the term “by weight” refers to the weight of a substance(for example, oleic acid, palmitic acid or PUFA such as linoleic acid orlinolenic acid) as a percentage of the weight of the compositioncomprising the substance or a component in the composition. For example,the weight of a particular fatty acid such as oleic acid may bedetermined as a percentage of the weight of the total fatty acid contentof the lipid or seedoil, or the seed.

As used herein, the term “biofuel” refers to any type of fuel, typicallyas used to power machinery such as automobiles, trucks or petroleumpowered motors, whose energy is derived from biological carbon fixationrather than from fossil fuel. Biofuels include fuels derived frombiomass conversion, as well as solid biomass, liquid fuels and biogases.Examples of biofuels include bioalcohols, biodiesel, synthetic diesel,vegetable oil, bioethers, biogas, syngas, solid biofuels, algae-derivedfuel, biohydrogen, biomethanol, 2,5-Dimethylfuran (DMF), biodimethylether (bioDME), Fischer-Tropsch diesel, biohydrogen diesel, mixedalcohols and wood diesel.

As used herein, the term “industrial product” refers to a hydrocarbonproduct which is predominantly made of carbon and hydrogen such as fattyacid methyl- and/or ethyl-esters or alkanes such as methane, mixtures oflonger chain alkanes which are typically liquids at ambienttemperatures, a biofuel, carbon monoxide and/or hydrogen, or abioalcohol such as ethanol, propanol, or butanol, or biochar. The term“industrial product” is intended to include intermediary products thatcan be converted to other industrial products, for example, syngas isitself considered to be an industrial product which can be used tosynthesize a hydrocarbon product which is also considered to be anindustrial product. The term industrial product as used herein includesboth pure forms of the above compounds, or more commonly a mixture ofvarious compounds and components, for example the hydrocarbon productmay contain a range of carbon chain lengths, as well understood in theart.

Polynucleotides

The terms “polynucleotide”, and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides. A polynucleotide of theinvention may be of genomic, cDNA, semisynthetic, or synthetic origin,double-stranded or single-stranded and by virtue of its origin ormanipulation: (1) is not associated with all or a portion of apolynucleotide with which it is associated in nature, (2) is linked to apolynucleotide other than that to which it is linked in nature, or (3)does not occur in nature. The following are non-limiting examples ofpolynucleotides: coding or non-coding regions of a gene or genefragment, loci (locus) defined from linkage analysis, exons, introns,messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA),ribozymes, cDNA, recombinant polynucleotides, plasmids, vectors,isolated DNA of any sequence, isolated RNA of any sequence, chimeric DNAof any sequence, nucleic acid probes, and primers. Preferredpolynucleotides of the invention include double-stranded DNA moleculeswhich are capable of being transcribed in plant cells and silencing RNAmolecules.

As used herein, the term “gene” is to be taken in its broadest contextand includes the deoxyribonucleotide sequences comprising thetranscribed region and, if translated, the protein coding region, of astructural gene and including sequences located adjacent to the codingregion on both the 5′ and 3′ ends for a distance of at least about 2 kbon either end and which are involved in expression of the gene. In thisregard, the gene includes control signals such as promoters, enhancers,termination and/or polyadenylation signals that are naturally associatedwith a given gene, or heterologous control signals, in which case, thegene is referred to as a “chimeric gene”. The sequences which arelocated 5′ of the protein coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the protein coding region and which arepresent on the mRNA are referred to as 3′ non-translated sequences. Theterm “gene” encompasses both cDNA and genomic forms of a gene. A genomicform or clone of a gene contains the coding region which may beinterrupted with non-coding sequences termed “introns”, “interveningregions”, or “intervening sequences.” Introns are segments of a genewhich are transcribed into nuclear RNA (nRNA). Introns may containregulatory elements such as enhancers. Introns are removed or “splicedout” from the nuclear or primary transcript; introns therefore areabsent in the mRNA transcript. The mRNA functions during translation tospecify the sequence or order of amino acids in a nascent polypeptide.The term “gene” includes a synthetic or fusion molecule encoding all orpart of the proteins of the invention described herein and acomplementary nucleotide sequence to any one of the above.

An “allele” refers to one specific form of a genetic sequence (such as agene) within a cell, an individual plant or within a population, thespecific form differing from other forms of the same gene in thesequence of at least one, and frequently more than one, variant siteswithin the sequence of the gene. The sequences at these variant sitesthat differ between different alleles are termed “variances”,“polymorphisms”, or “mutations”.

As used herein, “chimeric DNA” refers to any DNA molecule that is notnaturally found in nature; also referred to herein as a “DNA construct”.Typically, chimeric DNA comprises regulatory and transcribed or proteincoding sequences that are not naturally found together in nature.Accordingly, chimeric DNA may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that found in nature. The openreading frame may or may not be linked to its natural upstream anddownstream regulatory elements. The open reading frame may beincorporated into, for example, the plant genome, in a non-naturallocation, or in a replicon or vector where it is not naturally foundsuch as a bacterial plasmid or a viral vector. The term “chimeric DNA”is not limited to DNA molecules which are replicable in a host, butincludes DNA capable of being ligated into a replicon by, for example,specific adaptor sequences.

A “transgene” is a gene that has been introduced into the genome by atransformation procedure. The transgene may be in an initial transformedplant produced by regeneration from a transformed plant cell or inprogeny plants produced by self-fertilisation or crossing from theinitial transformant or in plant parts such as seeds. The term“genetically modified” and variations thereof include introducing a geneinto a cell by transformation or transduction, mutating a gene in a celland genetically altering or modulating the regulation of a gene in acell, or the progeny of any cell modified as described above.

A “genomic region” as used herein refers to a position within the genomewhere a transgene, or group of transgenes (also referred to herein as acluster), have been inserted into a cell, or predecessor thereof, suchthat they are co-inherited in progeny cells after meiosis.

A “recombinant polynucleotide” or “exogenous polynucleotide” of theinvention refers to a nucleic acid molecule which has been constructedor modified by artificial recombinant methods. The recombinantpolynucleotide may be present in a cell in an altered amount orexpressed at an altered rate (e.g., in the case of mRNA) compared to itsnative state. An exogenous polynucleotide is a polynucleotide that hasbeen introduced into a cell that does not naturally comprise thepolynucleotide. Typically an exogenous DNA is used as a template fortranscription of mRNA which is then translated into a continuoussequence of amino acid residues coding for a polypeptide of theinvention within the transformed cell. In another embodiment, part ofthe exogenous polynucleotide is endogenous to the cell and itsexpression is altered by recombinant means, for example, an exogenouscontrol sequence is introduced upstream of an endogenous polynucleotideto enable the transformed cell to express the polypeptide encoded by thepolynucleotide. For example, an exogenous polynucleotide may express anantisense RNA to an endogenous polynucleotide.

A recombinant polynucleotide of the invention includes polynucleotideswhich have not been separated from other components of the cell-based orcell-free expression system in which it is present, and polynucleotidesproduced in said cell-based or cell-free systems which are subsequentlypurified away from at least some other components. The polynucleotidecan be a contiguous stretch of nucleotides existing in nature, orcomprise two or more contiguous stretches of nucleotides from differentsources (naturally occurring and/or synthetic) joined to form a singlepolynucleotide. Typically, such chimeric polynucleotides comprise atleast an open reading frame encoding a polypeptide of the inventionoperably linked to a promoter suitable of driving transcription of theopen reading frame in a cell of interest.

With regard to the defined polynucleotides, it will be appreciated that% identity figures higher than those provided above will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polynucleotide comprises apolynucleotide sequence which is at least 50%, more preferably at least60%, more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99%, more preferably at least 99.1%, morepreferably at least 99.2%, more preferably at least 99.3%, morepreferably at least 99.4%, more preferably at least 99.5%, morepreferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

A polynucleotide of, or useful for, the present invention mayselectively hybridise, under stringent conditions, to a polynucleotidedefined herein. As used herein, stringent conditions are those that: (1)employ during hybridisation a denaturing agent such as formamide, forexample, 50% (v/v) formamide with 0.1% (w/v) bovine serum albumin, 0.1%Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or (2) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfateat 42° C. in 0.2×SSC and 0.1% SDS, and/or (3) employ low ionic strengthand high temperature for washing, for example, 0.015 M NaCl/0.0015 Msodium citrate/0.1% SDS at 50° C.

Polynucleotides of the invention may possess, when compared to naturallyoccurring molecules, one or more mutations which are deletions,insertions, or substitutions of nucleotide residues. Polynucleotideswhich have mutations relative to a reference sequence can be eithernaturally occurring (that is to say, isolated from a natural source) orsynthetic (for example, by performing site-directed mutagenesis or DNAshuffling on the nucleic acid as described above).

Polynucleotide for Reducing Expression Levels of Endogenous Proteins

In one embodiment, the cell/seed/plant/organism of the inventioncomprises an introduced mutation or an exogenous polynucleotide whichdown-regulates the production and/or activity of an endogenous enzyme,typically which results in an increased production of oleic acid, andpreferably a decreased production of palmitic acid and PUFA such aslinoleic acid, when compared to a corresponding cell lacking theintroduced mutation or exogenous polynucleotide. Examples of suchpolynucleotides include an antisense polynucleotide, a sensepolynucleotide, a catalytic polynucleotide, a microRNA, a polynucleotidewhich encodes a polypeptide which binds the endogenous enzyme and adouble stranded RNA.

RNA Interference

RNA interference (RNAi) is particularly useful for specificallyinhibiting the production of a particular protein. This technologyrelies on the presence of dsRNA molecules that contain a sequence thatis essentially identical to the mRNA of the gene of interest or partthereof and a sequence that is complementary thereto. Conveniently, thedsRNA can be produced from a single promoter in a recombinant vector orhost cell, where the sense and anti-sense sequences are covalentlyjoined by a sequence, preferably an unrelated sequence, which enablesthe sense and anti-sense sequences in the corresponding transcript tohybridize to form the dsRNA molecule with the joining sequence forming aloop structure, although a sequence with identity to the target RNA orits complement can form the loop structure. Typically, the dsRNA isencoded by a double-stranded DNA construct which has sense and antisensesequences in an inverted repeat structure, arranged as an interruptedpalindrome, where the repeated sequences are transcribed to produce thehybridising sequences in the dsRNA molecule, and the interrupt sequenceis transcribed to form the loop in the dsRNA molecule. The design andproduction of suitable dsRNA molecules is well within the capacity of aperson skilled in the art, particularly considering Waterhouse et al.(1998), Smith et al. (2000), WO 99/32619, WO 99/53050, WO 99/49029, andWO 01/34815.

In one example, a DNA is introduced that directs the synthesis of an atleast partly double stranded RNA product(s) with homology, preferably atleast 19 consecutive nucleotides complementary to a region of, a targetRNA, to be inactivated. The DNA therefore comprises both sense andantisense sequences that, when transcribed into RNA, can hybridize toform the double stranded RNA region. In one embodiment of the invention,the sense and antisense sequences are separated by a spacer region thatcomprises an intron which, when transcribed into RNA, is spliced out.This arrangement has been shown to result in a higher efficiency of genesilencing. The double stranded RNA region may comprise one or two RNAmolecules, transcribed from either one DNA region or two. The presenceof the double stranded molecule is thought to trigger a response from anendogenous system that destroys both the double stranded RNA and alsothe homologous RNA transcript from the target gene, efficiently reducingor eliminating the activity of the target gene.

The length of the sense and antisense sequences that hybridize shouldeach be at least 19 contiguous nucleotides, corresponding to part of thetarget mRNA. The full-length sequence corresponding to the entire genetranscript may be used. The degree of identity of the sense andantisense sequences to the targeted transcript should be at least 85%,at least 90%, or at least 95% to 100%. The RNA molecule may of coursecomprise unrelated sequences which may function to stabilize themolecule. The RNA molecule may be expressed under the control of a RNApolymerase II or RNA polymerase III promoter. Examples of the latterinclude tRNA or snRNA promoters.

Preferred small interfering RNA (“siRNA”) molecules comprise anucleotide sequence that is identical to about 19-21 contiguousnucleotides of the target mRNA. Preferably, the siRNA sequence commenceswith the dinucleotide AA, comprises a GC-content of about 30-70%(preferably, 30-60%, more preferably 40-60% and more preferably about45%-55%), and does not have a high percentage identity to any nucleotidesequence other than the target in the genome of the organism in which itis to be introduced, for example, as determined by standard BLASTsearch.

As an example, a dsRNA of the invention comprises a nucleotide sequenceprovided as any one of SEQ ID NOs 49 to 51 (where each T is a U).

microRNA

MicroRNAs (abbreviated miRNAs) are generally 19-25 nucleotides (commonlyabout 20-24 nucleotides in plants) non-coding RNA molecules that arederived from larger precursors that form imperfect stem-loop structures.

miRNAs bind to complementary sequences on target messenger RNAtranscripts (mRNAs), usually resulting in translational repression ortarget degradation and gene silencing.

In plant cells, miRNA precursor molecules are believed to be initiallyprocessed in the nucleus. The pri-miRNA (containing one or more localdouble-stranded or “hairpin” regions as well as the usual 5′ “cap” andpolyadenylated tail of an mRNA) is processed to a shorter miRNAprecursor molecule that also includes a stem-loop or fold-back structureand is termed the “pre-miRNA”. In plants, the pre-miRNAs are cleaved bydistinct DICER-like (DCL) enzymes, in particular DCL-1, yieldingmiRNA:miRNA* duplexes. Prior to transport out of the nucleus, theseduplexes are methylated. In contrast, hairpin RNA molecules havinglonger dsRNA regions are processed in particular by DCL-3 and DCL-4.

In the cytoplasm, the miRNA strand from the miRNA:miRNA duplex isselectively incorporated into an active RNA-induced silencing complex(RISC) for target recognition. The RISC-complexes contain a particularsubset of Argonaute proteins that exert sequence-specific generepression post-transcriptionally (see, for example, Millar andWaterhouse, 2005; Pasquinelli et al., 2005; Almeida and Allshire, 2005).

Cosuppression

Genes can suppress the expression of related endogenous genes and/ortransgenes already present in the genome, a phenomenon termedhomology-dependent gene silencing. Most of the instances of homologydependent gene silencing fall into two classes—those that function atthe level of transcription of the transgene, and those that operatepost-transcriptionally.

Post-transcriptional homology-dependent gene silencing (i.e.,cosuppression) describes the loss of expression of a transgene andrelated endogenous or viral genes in transgenic plants. Cosuppressionoften, but not always, occurs when transgene transcripts are abundant,and it is generally thought to be triggered at the level of mRNAprocessing, localization, and/or degradation. Several models exist toexplain how cosuppression works (see in Taylor, 1997).

Cosuppression involves introducing an extra copy of a gene or a fragmentthereof into a plant in the sense orientation with respect to a promoterfor its expression. A skilled person would appreciate that the size ofthe sense fragment, its correspondence to target gene regions, and itsdegree of sequence identity to the target gene can vary. In someinstances, the additional copy of the gene sequence interferes with theexpression of the target plant gene. Reference is made to WO 97/20936and EP 0465572 for methods of implementing co-suppression approaches.

Expression Vector

As used herein, an “expression vector” is a DNA or RNA vector that iscapable of transforming a host cell and of effecting expression of oneor more specified polynucleotides. Preferably, the expression vector isalso capable of replicating within the host cell or being integratedinto the host cell genome. Expression vectors are typically viruses orplasmids. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in host cells ofthe present invention, including in fungal, algal, and plant cells.

“Operably linked” as used herein, refers to a functional relationshipbetween two or more nucleic acid (e.g., DNA) segments. Typically, itrefers to the functional relationship of transcriptional regulatoryelement (promoter) to a transcribed sequence. For example, a promoter isoperably linked to a coding sequence of a polynucleotide defined herein,if it stimulates or modulates the transcription of the coding sequencein an appropriate cell. Generally, promoter transcriptional regulatoryelements that are operably linked to a transcribed sequence arephysically contiguous to the transcribed sequence, i.e., they arecis-acting. However, some transcriptional regulatory elements such asenhancers, need not be physically contiguous or located in closeproximity to the coding sequences whose transcription they enhance.

Expression vectors of the present invention contain regulatory sequencessuch as transcription control sequences, translation control sequences,origins of replication, and other regulatory sequences that arecompatible with the host cell and that control the expression ofpolynucleotides of the present invention. In particular, expressionvectors of the present invention include transcription controlsequences. Transcription control sequences are sequences which controlthe initiation, elongation, and termination of transcription.Particularly important transcription control sequences are those whichcontrol transcription initiation such as promoter, enhancer, operatorand repressor sequences. Suitable transcription control sequencesinclude any transcription control sequence that can function in at leastone of the recombinant cells of the present invention. The choice of theregulatory sequences used depends on the target organism such as a plantand/or target organ or tissue of interest. Such regulatory sequences maybe obtained from any eukaryotic organism such as plants or plantviruses, or may be chemically synthesized. A variety of suchtranscription control sequences are known to those skilled in the art.Particularly preferred transcription control sequences are promotersactive in directing transcription in plants, either constitutively orstage and/or tissue specific, depending on the use of the plant orpart(s) thereof

A number of vectors suitable for stable transfection of plant cells orfor the establishment of transgenic plants have been described in forexample, Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985,supp. 1987, Weissbach and Weissbach, Methods for Plant MolecularBiology, Academic Press, 1989, and Gelvin et al., Plant MolecularBiology Manual, Kluwer Academic Publishers, 1990. Typically, plantexpression vectors include for example, one or more cloned plant genesunder the transcriptional control of 5′ and 3′ regulatory sequences anda dominant selectable marker. Such plant expression vectors also cancontain a promoter regulatory region (e.g., a regulatory regioncontrolling inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific expression), atranscription initiation start site, a ribosome binding site, an RNAprocessing signal, a transcription termination site, and/or apolyadenylation signal.

A number of constitutive promoters that are active in plant cells havebeen described. Suitable promoters for constitutive expression in plantsinclude, but are not limited to, the cauliflower mosaic virus (CaMV) 35Spromoter, the Figwort mosaic virus (FMV) 35S, the sugarcane bacilliformvirus promoter, the commelina yellow mottle virus promoter, thelight-inducible promoter from the small subunit of theribulose-1,5-bis-phosphate carboxylase, the rice cytosolictriosephosphate isomerase promoter, the adeninephosphoribosyltransferase promoter of Arabidopsis, the rice actin 1 genepromoter, the mannopine synthase and octopine synthase promoters, theAdh promoter, the sucrose synthase promoter, the R gene complexpromoter, and the chlorophyll α/β binding protein gene promoter. Thesepromoters have been used to create DNA vectors that have been expressedin plants, see for example, WO 84/02913. All of these promoters havebeen used to create various types of plant-expressible recombinant DNAvectors.

For the purpose of expression in source tissues of the plant such as theleaf, seed, root or stem, it is preferred that the promoters utilized inthe present invention have relatively high expression in these specifictissues. For this purpose, one may choose from a number of promoters forgenes with tissue- or cell-specific, or -enhanced expression. Examplesof such promoters reported in the literature include, the chloroplastglutamine synthetase GS2 promoter from pea, the chloroplastfructose-1,6-biphosphatase promoter from wheat, the nuclearphotosynthetic ST-LS1 promoter from potato, the serine/threonine kinasepromoter and the glucoamylase (CHS) promoter from Arabidopsis thaliana.

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals, alsocan be used for expression of RNA-binding protein genes in plant cells,including promoters regulated by (1) heat, (2) light (e.g., pea RbcS-3Apromoter, maize RbcS promoter), (3) hormones such as abscisic acid, (4)wounding (e.g., WunI), or (5) chemicals such as methyl jasmonate,salicylic acid, steroid hormones, alcohol, Safeners (WO 97/06269), or itmay also be advantageous to employ (6) organ-specific promoters.

As used herein, the term “plant storage organ specific promoter” refersto a promoter that preferentially, when compared to other plant tissues,directs gene transcription in a storage organ of a plant. The plantstorage organ is preferably a seed. Preferably, the promoter onlydirects expression of a gene of interest in the storage organ, and/orexpression of the gene of interest in other parts of the plant such asleaves is not detectable by Northern blot analysis and/or RT-PCR.Typically, the promoter drives expression of genes during growth anddevelopment of the storage organ, in particular during the phase ofsynthesis and accumulation of storage compounds in the storage organ.Such promoters may drive gene expression in the entire plant storageorgan or only part thereof such as the seedcoat, embryo or cotyledon(s)in seeds of dicotyledonous plants or the endosperm or aleurone layer ofseeds of monocotyledonous plants.

Other promoters can also be used to express a protein in specifictissues such as seeds or fruits. The promoter for β-conglycinin or otherseed-specific promoters such as the napin, zein, linin and phaseolinpromoters, can be used. In one embodiment, the promoter directsexpression in tissues and organs in which lipid biosynthesis take place.Such promoters act in seed development at a suitable time for modifyinglipid composition in seeds. In one embodiment, the plant storage organspecific promoter is a seed specific promoter. In a more preferredembodiment, the promoter preferentially directs expression in the embryoand/or cotyledons of a dicotyledonous plant or in the endosperm of amonocotyledonous plant, relative to expression in other organs in theplant such as leaves. Preferred promoters for seed-specific expressioninclude: 1) promoters from genes encoding enzymes involved in lipidbiosynthesis and accumulation in seeds such as desaturases andelongases, 2) promoters from genes encoding seed storage proteins, and3) promoters from genes encoding enzymes involved in carbohydratebiosynthesis and accumulation in seeds. Seed specific promoters whichare suitable are, a flax linin promoter (e.g. Cnl1 or Cnl2 promoters)the oilseed rape napin gene promoter (U.S. Pat. No. 5,608,152), theVicia faba USP promoter (Baumlein et al., 1991), the Arabidopsis oleosinpromoter (WO 98/45461), the Phaseolus vulgaris phaseolin promoter (U.S.Pat. No. 5,504,200), the Brassica Bce4 promoter (WO 91/13980), or thelegumin B4 promoter (Baumlein et al., 1992), and promoters which lead tothe seed-specific expression in monocots such as maize, barley, wheat,rye, rice and the like. Notable promoters which are suitable are thebarley lpt2 or lpt1 gene promoter (WO 95/15389 and WO 95/23230), or thepromoters described in WO 99/16890 (promoters from the barley hordeingene, the rice glutelin gene, the rice oryzin gene, the rice prolamingene, the wheat gliadin gene, the wheat glutelin gene, the maize zeingene, the oat glutelin gene, the sorghum kasirin gene, the rye secalingene). Other promoters include those described by Broun et al. (1998),Potenza et al. (2004), US 20070192902 and US 20030159173. In anembodiment, the seed specific promoter is preferentially expressed indefined parts of the seed such as the embryo, cotyledon(s) or theendosperm. Examples of cotyledon specific promoters include, but are notlimited to, the FP1 promoter (Ellerstrom et al., 1996), the pea leguminpromoter (Perrin et al., 2000), and the bean phytohemagglutnin promoter(Perrin et al., 2000). In a further embodiment, the seed specificpromoter is not expressed, or is only expressed at a low level, in theembryo and/or after the seed germinates.

When there are multiple promoters present, each promoter mayindependently be the same or different.

The 5′ non-translated leader sequence can be derived from the promoterselected to express the heterologous gene sequence of thepolynucleotide, or may be heterologous with respect to the coding regionof the enzyme to be produced, and can be specifically modified ifdesired so as to increase translation of mRNA.

The termination of transcription is accomplished by a 3′ non-translatedDNA sequence operably linked in the expression vector to thepolynucleotide of interest.

The 3′ non-translated region of a recombinant DNA molecule contains apolyadenylation signal that functions in plants to cause the addition ofadenylate nucleotides to the 3′ end of the RNA. The 3′ non-translatedregion can be obtained from various genes that are expressed in, forexample, plant cells. The nopaline synthase 3′ untranslated region, the3′ untranslated region from pea small subunit Rubisco gene, the 3′untranslated region from soybean 7S seed storage protein gene arecommonly used in this capacity. The 3′ transcribed, non-translatedregions containing the polyadenylate signal of Agrobacteriumtumor-inducing (Ti) plasmid genes are also suitable.

Recombinant DNA technologies can be used to improve expression of atransformed polynucleotide by manipulating for example, the number ofcopies of the polynucleotide within a host cell, the efficiency withwhich those polynucleotide are transcribed, the efficiency with whichthe resultant transcripts are translated, and the efficiency ofpost-translational modifications.

To facilitate identification of transformants, the recombinant vectordesirably comprises a selectable or screenable marker gene as, or inaddition to, the nucleic acid sequence of a polynucleotide definedherein. By “marker gene” is meant a gene that imparts a distinctphenotype to cells expressing the marker gene and thus, allows suchtransformed cells to be distinguished from cells that do not have themarker. A selectable marker gene confers a trait for which one can“select” based on resistance to a selective agent (e.g., a herbicide,antibiotic, radiation, heat, or other treatment damaging tountransformed cells). A screenable marker gene (or reporter gene)confers a trait that one can identify through observation or testing,that is, by “screening” (e.g., β-glucuronidase, luciferase, GFP or otherenzyme activity not present in untransformed cells). The marker gene andthe nucleotide sequence of interest do not have to be linked, sinceco-transformation of unlinked genes as for example, described in U.S.Pat. No. 4,399,216, is also an efficient process in for example, planttransformation. The actual choice of a marker is not crucial as long asit is functional (i.e., selective) in combination with the cells ofchoice such as a plant cell.

Exemplary selectable markers for selection of plant transformantsinclude, but are not limited to, a hyg gene which encodes hygromycin Bresistance; a neomycin phosphotransferase (nptII) gene conferringresistance to kanamycin, paromomycin, G418; a glutathione-S-transferasegene from rat liver conferring resistance to glutathione derivedherbicides as for example, described in EP 256223; a glutaminesynthetase gene conferring, upon overexpression, resistance to glutaminesynthetase inhibitors such as phosphinothricin as for example, describedin WO 87/05327; an acetyltransferase gene from Streptomycesviridochromogenes conferring resistance to the selective agentphosphinothricin as for example, described in EP 275957; a gene encodinga 5-enolshikimate-3-phosphate synthase (EPSPS) conferring tolerance toN-phosphonomethylglycine as for example, described by Hinchee et al.(1988); a bar gene conferring resistance against bialaphos as forexample, described in WO91/02071; a nitrilase gene such as bxn fromKlebsiella ozaenae which confers resistance to bromoxynil (Stalker etal., 1988); a dihydrofolate reductase (DHFR) gene conferring resistanceto methotrexate (Thillet et al., 1988); a mutant acetolactate synthasegene (ALS) which confers resistance to imidazolinone, sulfonylurea, orother ALS-inhibiting chemicals (EP 154,204); a mutated anthranilatesynthase gene that confers resistance to 5-methyl tryptophan; or adalapon dehalogenase gene that confers resistance to the herbicide.

Preferred screenable markers include, but are not limited to, a uidAgene encoding a β-glucuronidase (GUS) enzyme for which variouschromogenic substrates are known; a β-galactosidase gene encoding anenzyme for which chromogenic substrates are known; an aequorin gene(Prasher et al., 1985) which may be employed in calcium-sensitivebioluminescence detection; a green fluorescent protein gene (Niedz etal., 1995) or derivatives thereof; or a luciferase (luc) gene (Ow etal., 1986) which allows for bioluminescence detection. By “reportermolecule” it is meant a molecule that, by its chemical nature, providesan analytically identifiable signal that facilitates determination ofpromoter activity by reference to protein product.

Preferably, the recombinant vector is stably incorporated into thegenome of the cell such as the plant cell. Accordingly, the recombinantvector may comprise appropriate elements which allow the vector to beincorporated into the genome, or into a chromosome of the cell.

Transfer Nucleic Acids

Transfer nucleic acids can be used to deliver an exogenouspolynucleotide to a cell and comprise one, preferably two, bordersequences and a polynucleotide of interest. The transfer nucleic acidmay or may not encode a selectable marker. Preferably, the transfernucleic acid forms part of a binary vector in a bacterium, where thebinary vector further comprises elements which allow replication of thevector in the bacterium, selection, or maintenance of bacterial cellscontaining the binary vector. Upon transfer to a eukaryotic cell, thetransfer nucleic acid component of the binary vector is capable ofintegration into the genome of the eukaryotic cell.

As used herein, the term “extrachromosomal transfer nucleic acid” refersto a nucleic acid molecule that is capable of being transferred from abacterium such as Agrobacterium sp., to a eukaryotic cell such as aplant cell. An extrachromosomal transfer nucleic acid is a geneticelement that is well-known as an element capable of being transferred,with the subsequent integration of a nucleotide sequence containedwithin its borders into the genome of the recipient cell. In thisrespect, a transfer nucleic acid is flanked, typically, by two “border”sequences, although in some instances a single border at one end can beused and the second end of the transferred nucleic acid is generatedrandomly in the transfer process. A polynucleotide of interest istypically positioned between the left border-like sequence and the rightborder-like sequence of a transfer nucleic acid. The polynucleotidecontained within the transfer nucleic acid may be operably linked to avariety of different promoter and terminator regulatory elements thatfacilitate its expression, that is, transcription and/or translation ofthe polynucleotide. Transfer DNAs (T-DNAs) from Agrobacterium sp. suchas Agrobacterium tumefaciens or Agrobacterium rhizogenes, and man madevariants/mutants thereof are probably the best characterized examples oftransfer nucleic acids. Another example is P-DNA (“plant-DNA”) whichcomprises T-DNA border-like sequences from plants.

As used herein, “T-DNA” refers to for example, T-DNA of an Agrobacteriumtumefaciens Ti plasmid or from an Agrobacterium rhizogenes Ri plasmid,or man made variants thereof which function as T-DNA. The T-DNA maycomprise an entire T-DNA including both right and left border sequences,but need only comprise the minimal sequences required in cis fortransfer, that is, the right and T-DNA border sequence. The T-DNAs ofthe invention have inserted into them, anywhere between the right andleft border sequences (if present), the polynucleotide of interest. Thesequences encoding factors required in trans for transfer of the T-DNAinto a plant cell such as vir genes, may be inserted into the T-DNA, ormay be present on the same replicon as the T-DNA, or preferably are intrans on a compatible replicon in the Agrobacterium host. Such “binaryvector systems” are well known in the art.

As used herein, “P-DNA” refers to a transfer nucleic acid isolated froma plant genome, or man made variants/mutants thereof, and comprises ateach end, or at only one end, a T-DNA border-like sequence. Theborder-like sequence preferably shares at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 90%, or at least 95%,but less than 100% sequence identity, with a T-DNA border sequence froman Agrobacterium sp. such as Agrobacterium tumefaciens or Agrobacteriumrhizogenes. Thus, P-DNAs can be used instead of T-DNAs to transfer anucleotide sequence contained within the P-DNA from, for exampleAgrobacterium, to another cell. The P-DNA, before insertion of theexogenous polynucleotide which is to be transferred, may be modified tofacilitate cloning and should preferably not encode any proteins. TheP-DNA is characterized in that it contains, at least a right bordersequence and preferably also a left border sequence.

As used herein, a “border” sequence of a transfer nucleic acid can beisolated from a selected organism such as a plant or bacterium, or be aman made variant/mutant thereof. The border sequence promotes andfacilitates the transfer of the polynucleotide to which it is linked andmay facilitate its integration in the recipient cell genome. In anembodiment, a border-sequence is between 5-100 base pairs (bp) inlength, 10-80 bp in length, 15-75 bp in length, 15-60 bp in length,15-50 bp in length, 15-40 bp in length, 15-30 bp in length, 16-30 bp inlength, 20-30 bp in length, 21-30 bp in length, 22-30 bp in length,23-30 bp in length, 24-30 bp in length, 25-30 bp in length, or 26-30 bpin length. Border sequences from T-DNA from Agrobacterium sp. are wellknown in the art and include those described in Lacroix et al. (2008),Tzfira and Citovsky (2006) and Glevin (2003).

Whilst traditionally only Agrobacterium sp. have been used to transfergenes to plants cells, there are now a large number of systems whichhave been identified/developed which act in a similar manner toAgrobacterium sp. Several non-Agrobacterium species have recently beengenetically modified to be competent for gene transfer (Chung et al.,2006; Broothaerts et al., 2005). These include Rhizobium sp. NGR234,Sinorhizobium meliloti and Mezorhizobium loti. The bacteria are madecompetent for gene transfer by providing the bacteria with the machineryneeded for the transformation process, that is, a set of virulence genesencoded by an Agrobacterium Ti-plasmid and the T-DNA segment residing ona separate, small binary plasmid. Bacteria engineered in this way arecapable of transforming different plant tissues (leaf disks, calli andoval tissue), monocots or dicots, and various different plant species(e.g., tobacco, rice).

As used herein, the terms “transfection”, “transformation” andvariations thereof are generally used interchangeably. “Transfected” or“transformed” cells may have been manipulated to introduce thepolynucleotide(s) of interest, or may be progeny cells derivedtherefrom.

Recombinant Cells

The invention also provides a recombinant cell, for example, arecombinant plant cell, which is a host cell transformed with one ormore polynucleotides or vectors defined herein, or combination thereof.The term “recombinant cell” is used interchangeably with the term“transgenic cell” herein. Suitable cells of the invention include anycell that can be transformed with a polynucleotide or recombinant vectorof the invention, encoding for example, a polypeptide or enzymedescribed herein. The cell is preferably a cell which is thereby capableof being used for producing lipid. The recombinant cell may be a cell inculture, a cell in vitro, or in an organism such as for example, aplant, or in an organ such as, for example, a seed or a leaf.Preferably, the cell is in a plant, more preferably in the seed of aplant, more preferably in the seed of an oilseed plant such assafflower. In an embodiment, the plant cell comprises lipid or oilhaving the fatty acid composition as described herein.

Host cells into which the polynucleotide(s) are introduced can be eitheruntransformed cells or cells that are already transformed with at leastone nucleic acid. Such nucleic acids may be related to lipid synthesis,or unrelated. Host cells of the present invention either can beendogenously (i.e., naturally) capable of producing polypeptide(s)defined herein, in which case the recombinant cell derived therefrom hasan enhanced capability of producing the polypeptide(s), or can becapable of producing said polypeptide(s) only after being transformedwith at least one polynucleotide of the invention. In an embodiment, arecombinant cell of the invention has an enhanced capacity to producenon-polar lipid. The cells may be prokaryotic or eukaryotic. Preferredhost cells are yeast, algal and plant cells. In a preferred embodiment,the plant cell is a seed cell, in particular, a cell in a cotyledon orendosperm of a seed. Examples of algal cells useful as host cells of thepresent invention include, for example, Chlamydomonas sp. (for example,Chiamydomonas reinhardtii), Dunaliella sp., Haematococcus sp., Chlorellasp., Thraustochytrium sp., Schizochytrium sp., and Volvox sp.

The host cells may be of an organism suitable for a fermentationprocess, such as, for example, Yarrowia lipolytica or other yeasts.

Transgenic Plants

The invention also provides a plant comprising an exogenouspolynucleotide or polypeptide of the invention, a cell of the invention,a vector of the invention, or a combination thereof. The term “plant”refers to whole plants, whilst the term “part thereof” refers to plantorgans (e.g., leaves, stems, roots, flowers, fruit), single cells (e.g.,pollen), seed, seed parts such as an embryo, endosperm, scutellum orseed coat, plant tissue such as vascular tissue, plant cells and progenyof the same. As used herein, plant parts comprise plant cells.

As used herein, the term “plant” is used in it broadest sense. Itincludes, but is not limited to, any species of grass, ornamental ordecorative plant, crop or cereal (e.g., oilseed plants, maize, soybean),fodder or forage, fruit or vegetable plant, herb plant, woody plant,flower plant, or tree. It is not meant to limit a plant to anyparticular structure. It also refers to a unicellular plant (e.g.,microalga). The term “part thereof” in reference to a plant refers to aplant cell and progeny of same, a plurality of plant cells that arelargely differentiated into a colony (e.g., volvox), a structure that ispresent at any stage of a plant's development, or a plant tissue. Suchstructures include, but are not limited to, leaves, stems, flowers,fruits, nuts, roots, seed, seed coat, embryos. The term “plant tissue”includes differentiated and undifferentiated tissues of plants includingthose present in leaves, stems, flowers, fruits, nuts, roots, seed, forexample, embryonic tissue, endosperm, dermal tissue (e.g., epidermis,periderm), vascular tissue (e.g., xylem, phloem), or ground tissue(comprising parenchyma, collenchyma, and/or sclerenchyma cells), as wellas cells in culture (e.g., single cells, protoplasts, callus, embryos,etc.). Plant tissue may be in planta, in organ culture, tissue culture,or cell culture.

A “transgenic plant”, “genetically modified plant” or variations thereofrefers to a plant that contains a transgene not found in a wild-typeplant of the same species, variety or cultivar. Transgenic plants asdefined in the context of the present invention include plants and theirprogeny which have been genetically modified using recombinanttechniques to cause production of at least one polypeptide definedherein in the desired plant or part thereof “Transgenic plant parts” hasa corresponding meaning.

The terms “seed” and “grain” are related terms as used herein, and haveoverlapping meanings. “Grain” refers to mature grain such as harvestedgrain or grain which is still on a plant but ready for harvesting, butcan also refer to grain after imbibition or germination, according tothe context. Mature grain commonly has a moisture content of less thanabout 18-20%. “Seed” includes “developing seed” as well as “grain” whichis mature grain, but not grain after imbibition or germination.“Developing seed” as used herein refers to a seed prior to maturity,typically found in the reproductive structures of the plant afterfertilisation or anthesis, but can also refer to such seeds prior tomaturity which are isolated from a plant. Seed development in planta istypically divided into early-, mid-, and late phases of development.

As used herein, the term “plant storage organ” refers to a part of aplant specialized to store energy in the form of for example, proteins,carbohydrates, lipid. Examples of plant storage organs are seed, fruit,tuberous roots, and tubers. A preferred plant storage organ of theinvention is seed.

As used herein, the term “vegetative tissue” or “vegetative plant part”or variants thereof is any plant tissue, organ or part that does notinclude the organs for sexual reproduction of plants or the seed bearingorgans or the closely associated tissues or organs such as flowers,fruits and seeds. Vegetative tissues and parts include at least plantleaves, stems (including bolts and tillers but excluding the heads),tubers and roots, but excludes flowers, pollen, seed including the seedcoat, embryo and endosperm, fruit including mesocarp tissue,seed-bearing pods and seed-bearing heads. In one embodiment, thevegetative part of the plant is an aerial plant part. In another orfurther embodiment, the vegetative plant part is a green part such as aleaf or stem. Vegetative parts include those parts principally involvedin providing or supporting the photosynthetic capacity of the plant orrelated function, or anchoring the plant.

As used herein, the term “phenotypically normal” refers to a geneticallymodified plant or part thereof, particularly a storage organ such as aseed of the invention not having a significantly reduced ability to growand reproduce when compared to an unmodified plant or plant thereof. Inan embodiment, the genetically modified plant or part thereof which isphenotypically normal comprises at least one exogenous polynucleotide asdefined herein and has an ability to grow or reproduce which isessentially the same as a corresponding plant or part thereof notcomprising said polynucleotide(s). Preferably, the biomass, growth rate,germination rate, storage organ size, seed size and/or the number ofviable seeds produced is not less than 90% of that of a plant lackingsaid exogenous polynucleotide when grown under identical conditions.This term does not encompass features of the plant which may bedifferent to the wild-type plant but which do not effect the usefulnessof the plant for commercial purposes.

Plants provided by or contemplated for use in the practice of thepresent invention include both monocotyledons and dicotyledons. Inpreferred embodiments, the plants of the present invention are cropplants (for example, cereals and pulses, maize, wheat, potatoes,tapioca, rice, sorghum, millet, cassava, barley, or pea), or otherlegumes. The plants may be grown for production of edible roots, tubers,leaves, stems, flowers or fruit. The plants may be vegetable orornamental plants. The plants of the invention may be: safflower(Carthamus tinctorius), corn (Zea mays), canola (Brassica napus,Brassica rapa ssp.), other Brassicas such as, for example, rutabaga(Brassica napobrassica), mustard (Brassica juncea), Ethiopian mustard(Brassica carinata), crambe (Crambe abyssinica), camelina (Camelinasativa), sugarbeet (Beta vulgaris), clover (Trifolium sp.), flax (Linumusitatissimum), alfalfa (Medicago sativa), rice (Oryza sativa), rye(Secale cerale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower(Helianthus annus), wheat (Tritium aestivum), soybean (Glycine max),tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Lopmoeabatatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut(Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus spp.),cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.),avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava),mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardiurn occidentale), macadamia (Macadamia intergrifolia),almond (Prunus amygdalus), jatropha (Jatropha curcas), lupins,Eucalypts, palm, nut sage, pongamia, oats, or barley.

Other preferred plants include C4 grasses such as Andropogon gerardi,Bouteloua curtipendula, B. gracilis, Buchloe dactyloides, Panicumvirgatum, Schizachyrium scoparium, Miscanthus species for example,Miscanthus x giganteus and Miscanthus sinensis, Sorghastrum nutans,Sporobolus cryptandrus, Switchgrass (Panicum virgatum), sugarcane(Saccharum officinarum), Brachyaria; C3 grasses such as Elymuscanadensis, the legumes Lespedeza capitata and Petalostemum villosum,the forb Aster azureus; and woody plants such as Quercus ellipsoidalisand Q. macrocarpa.

In a preferred embodiment, the plant is an angiosperm.

In a preferred embodiment, the plant is an oilseed plant, preferably anoilseed crop plant. As used herein, an “oilseed plant” is a plantspecies used for the commercial production of lipid from the seeds ofthe plant. “Commercial production” herein means the production of lipid,preferably oil, for sale in return for revenue. The oilseed plant may beoil-seed rape (such as canola), maize, sunflower, safflower, soybean,sorghum, flax (linseed) or sugar beet. Furthermore, the oilseed plantmay be other Brassicas, cotton, peanut, poppy, rutabaga, mustard, castorbean, sesame, safflower, or nut producing plants. The plant may producehigh levels of lipid in its fruit such as olive, oil palm or coconut.Horticultural plants to which the present invention may be applied arelettuce, endive, or vegetable Brassicas including cabbage, broccoli, orcauliflower. The present invention may be applied in tobacco, cucurbits,carrot, strawberry, tomato, or pepper. More preferred plants are oilseedplants whose developing seeds are non-photosynthetic, also referred toas “white seeds” plants, such as safflower, sunflower, cotton andcastor.

In a preferred embodiment, the transgenic plant is homozygous for eachand every gene that has been introduced (transgene) so that its progenydo not segregate for the desired phenotype. The transgenic plant mayalso be heterozygous for the introduced transgene(s), preferablyuniformly heterozygous for the transgene such as for example, in F1progeny which have been grown from hybrid seed. Such plants may provideadvantages such as hybrid vigour, well known in the art.

Transformation of Plants

Transgenic plants can be produced using techniques known in the art,such as those generally described in Slater et al., PlantBiotechnology—The Genetic Manipulation of Plants, Oxford UniversityPress (2003), and Christou and Klee, Handbook of Plant Biotechnology,John Wiley and Sons (2004).

As used herein, the terms “stably transforming”, “stably transformed”and variations thereof refer to the integration of the polynucleotideinto the genome of the cell such that they are transferred to progenycells during cell division without the need for positively selecting fortheir presence. Stable transformants, or progeny thereof, can beselected and/or identified by any means known in the art such asSouthern blots on chromosomal DNA, or in situ hybridization of genomicDNA.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because DNA can be introduced intocells in whole plant tissues, plant organs, or explants in tissueculture, for either transient expression, or for stable integration ofthe DNA in the plant cell genome. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (see for example, U.S. Pat. Nos. 5,177,010, 5,104,310,5,004,863, or U.S. Pat. No. 5,159,135). The region of DNA to betransferred is defined by the border sequences, and the intervening DNA(T-DNA) is usually inserted into the plant genome. Further, theintegration of the T-DNA is a relatively precise process resulting infew rearrangements. In those plant varieties whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.Preferred Agrobacterium transformation vectors are capable ofreplication in E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., In: Plant DNA InfectiousAgents, Hohn and Schell, eds., Springer-Verlag, New York, pp. 179-203(1985)).

Acceleration methods that may be used include for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang etal., Particle Bombardment Technology for Gene Transfer, Oxford Press,Oxford, England (1994). Non-biological particles (microprojectiles) thatmay be coated with nucleic acids and delivered into cells by apropelling force. Such methods are well known in the art. In anotherembodiment, plastids can be stably transformed. Methods disclosed forplastid transformation in higher plants include particle gun delivery ofDNA containing a selectable marker and targeting of the DNA to theplastid genome through homologous recombination (U.S. Pat. Nos.5,451,513, 5,545,818, 5,877,402, 5,932,479, and WO 99/05265).

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments. Application ofthese systems to different plant varieties depends upon the ability toregenerate that particular plant strain from protoplasts. Illustrativemethods for the regeneration of cereals from protoplasts are described(Fujimura et al., 1985; Toriyama et al., 1986; Abdullah et al., 1986).Other methods of cell transformation can also be used and include butare not limited to the introduction of DNA into plants by direct DNAtransfer into pollen, by direct injection of DNA into reproductiveorgans of a plant, or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos.

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach et al., In: Methods for Plant MolecularBiology, Academic Press, San Diego, Calif., (1988)). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene is well known in the art. Preferably, the regeneratedplants are self-pollinated to provide homozygous transgenic plants.Otherwise, pollen obtained from the regenerated plants is crossed toseed-grown plants of agronomically important lines. Conversely, pollenfrom plants of these important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolynucleotide is cultivated using methods well known to one skilled inthe art.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens, and obtaining transgenic plants have been published forcotton (U.S. Pat. Nos. 5,004,863, 5,159,135, 5,518,908), soybean (U.S.Pat. Nos. 5,569,834, 5,416,011), Brassica (U.S. Pat. No. 5,463,174),peanut (Cheng et al., 1996), and pea (Grant et al., 1995).

Methods for transformation of cereal plants such as wheat and barley forintroducing genetic variation into the plant by introduction of anexogenous nucleic acid and for regeneration of plants from protoplastsor immature plant embryos are well known in the art, see for example, CA2,092,588, AU 61781/94, AU 667939, U.S. Pat. No. 6,100,447,PCT/US97/10621, U.S. Pat. Nos. 5,589,617, 6,541,257. The regenerablewheat cells are preferably from the scutellum of immature embryos,mature embryos, callus derived from these, or the meristematic tissue.

To confirm the presence of the transgenes in transgenic cells andplants, a polymerase chain reaction (PCR) amplification or Southern blotanalysis can be performed using methods known to those skilled in theart. Once transgenic plants have been obtained, they may be grown toproduce plant tissues or parts having the desired phenotype. The planttissue or plant parts, may be harvested, and/or the seed collected. Theseed may serve as a source for growing additional plants with tissues orparts having the desired characteristics.

A transgenic plant formed using Agrobacterium or other transformationmethods typically contains a single transgenic locus on one chromosome.Such transgenic plants can be referred to as being hemizygous for theadded gene(s). More preferred is a transgenic plant that is homozygousfor the added gene(s), that is, a transgenic plant that contains twoadded genes, one gene at the same locus on each chromosome of achromosome pair. A homozygous transgenic plant can be obtained byself-fertilising a hemizygous transgenic plant, germinating some of theseed produced and analyzing the resulting plants for the gene ofinterest.

It is also to be understood that two different transgenic plants thatcontain two independently segregating exogenous genes or loci can alsobe crossed (mated) to produce offspring that contain both sets of genesor loci. Selfing of appropriate F1 progeny can produce plants that arehomozygous for both exogenous genes or loci. Back-crossing to a parentalplant and out-crossing with a non-transgenic plant are alsocontemplated, as is vegetative propagation. Descriptions of otherbreeding methods that are commonly used for different traits and cropscan be found in Fehr, In: Breeding Methods for Cultivar Development,Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987).

For the transformation of safflower, particularly useful methods aredescribed by Belide et al. (2011).

Tilling

In one embodiment, TILLING (Targeting Induced Local Lesions IN Genomes)can be used to produce plants in which endogenous genes are knocked out,for example genes encoding a Δ12 desaturase, a palmitoyl-ACPthioesterase, a ω6 or a Δ6 desaturase activity, or a combination of twoor more thereof.

In a first step, introduced mutations such as novel single base pairchanges are induced in a population of plants by treating seeds (orpollen) with a chemical mutagen, and then advancing plants to ageneration where mutations will be stably inherited. DNA is extracted,and seeds are stored from all members of the population to create aresource that can be accessed repeatedly over time.

For a TILLING assay, PCR primers are designed to specifically amplify asingle gene target of interest. Specificity is especially important if atarget is a member of a gene family or part of a polyploid genome. Next,dye-labeled primers can be used to amplify PCR products from pooled DNAof multiple individuals. These PCR products are denatured and reannealedto allow the formation of mismatched base pairs. Mismatches, orheteroduplexes, represent both naturally occurring single nucleotidepolymorphisms (SNPs) (i.e., several plants from the population arelikely to carry the same polymorphism) and induced SNPs (i.e., only rareindividual plants are likely to display the mutation). Afterheteroduplex formation, the use of an endonuclease, such as Cell, thatrecognizes and cleaves mismatched DNA is the key to discovering novelSNPs within a TILLING population.

Using this approach, many thousands of plants can be screened toidentify any individual with a single base change as well as smallinsertions or deletions (1-30 bp) in any gene or specific region of thegenome. Genomic fragments being assayed can range in size anywhere from0.3 to 1.6 kb. At 8-fold pooling, 1.4 kb fragments (discounting the endsof fragments where SNP detection is problematic due to noise) and 96lanes per assay, this combination allows up to a million base pairs ofgenomic DNA to be screened per single assay, making TILLING ahigh-throughput technique. TILLING is further described in Slade andKnauf (2005), and Henikoff et al. (2004).

In addition to allowing efficient detection of mutations,high-throughput TILLING technology is ideal for the detection of naturalpolymorphisms. Therefore, interrogating an unknown homologous DNA byheteroduplexing to a known sequence reveals the number and position ofpolymorphic sites. Both nucleotide changes and small insertions anddeletions are identified, including at least some repeat numberpolymorphisms. This has been called Ecotilling (Comai et al., 2004).

Each SNP is recorded by its approximate position within a fewnucleotides. Thus, each haplotype can be archived based on its mobility.Sequence data can be obtained with a relatively small incremental effortusing aliquots of the same amplified DNA that is used for themismatch-cleavage assay. The left or right sequencing primer for asingle reaction is chosen by its proximity to the polymorphism.Sequencher software performs a multiple alignment and discovers the basechange, which in each case confirmed the gel band.

Ecotilling can be performed more cheaply than full sequencing, themethod currently used for most SNP discovery. Plates containing arrayedecotypic DNA can be screened rather than pools of DNA from mutagenizedplants. Because detection is on gels with nearly base pair resolutionand background patterns are uniform across lanes, bands that are ofidentical size can be matched, thus discovering and genotyping SNPs in asingle step. In this way, ultimate sequencing of the SNP is simple andefficient, made more so by the fact that the aliquots of the same PCRproducts used for screening can be subjected to DNA sequencing.

Mutagenesis Procedures

Techniques for generating mutant plant lines are known in the art.Examples of mutagens that can be used for generating mutant plantsinclude irradiation and chemical mutagenesis. Mutants may also beproduced by techniques such as T-DNA insertion and transposon-inducedmutagenesis. Mutations in any desired gene in a plant, can be introducedin a site-specific manner by artificial zinc finger nuclease (ZFN), TALeffector (TALEN) or CRISPR technologies (using a Cas9 type nuclease) asknown in the art. The mutagenesis procedure may be performed on anyparental cell of a plant, for example a seed or a parental cell intissue culture.

Chemical mutagens are classifiable by chemical properties, e.g.,alkylating agents, cross-linking agents, etc. Useful chemical mutagensinclude, but are not limited to, N-ethyl-N-nitrosourea (ENU);N-methyl-N-nitrosourea (MNU); procarbazine hydrochloride; chlorambucil;cyclophosphamide; methyl methanesulfonate (MMS); ethyl methanesulfonate(EMS); diethyl sulfate; acrylamide monomer; triethylene melamine (TEM);melphalan; nitrogen mustard; vincristine; dimethylnitrosamine;N-methyl-N′-nitro-Nitrosoguani-dine (MNNG); 7,12 dimethylbenzanthracene(DMBA); ethylene oxide; hexamethylphosphoramide; and bisulfan.

An example of suitable irradiation to induce mutations is by gammaradiation, such as that supplied by a Cesium 137 source. The gammaradiation preferably is supplied to the plant cells in a dosage ofapproximately 60 to 200 Krad., and most preferably in a dosage ofapproximately 60 to 90 Krad.

Plants are typically exposed to a mutagen for a sufficient duration toaccomplish the desired genetic modification but insufficient tocompletely destroy the viability of the cells and their ability to beregenerated into a plant.

The mutagenesis procedures described above typically result in thegeneration of mutants in a gene of interest at a frequency of at least 1in 1000 plants, which means that screening of mutagenised populations ofthe plants is a practicable means to identify mutants in any gene ofinterest. The identification of mutants can also be achieved bymassively parallel nucleotide sequencing technologies.

Marker Assisted Selection

Marker assisted selection is a well recognised method of selecting forheterozygous plants required when backcrossing with a recurrent parentin a classical breeding program. The population of plants in eachbackcross generation will be heterozygous for the gene of interestnormally present in a 1:1 ratio in a backcross population, and themolecular marker can be used to distinguish the two alleles of the gene.By extracting DNA from, for example, young shoots and testing with aspecific marker for the introgressed desirable trait, early selection ofplants for further backcrossing is made whilst energy and resources areconcentrated on fewer plants. To further speed up the backcrossingprogram, the embryo from immature seeds (25 days post anthesis) may beexcised and grown up on nutrient media under sterile conditions, ratherthan allowing full seed maturity. This process, termed “embryo rescue”,used in combination with DNA extraction at the three leaf stage andanalysis for the desired genotype allows rapid selection of plantscarrying the desired trait, which may be nurtured to maturity in thegreenhouse or field for subsequent further backcrossing to the recurrentparent.

Any molecular biological technique known in the art which is capable ofdetecting a FAD2, FATB or FAD6 gene can be used in the methods of thepresent invention. Such methods include, but are not limited to, the useof nucleic acid amplification, nucleic acid sequencing, nucleic acidhybridization with suitably labeled probes, single-strand conformationalanalysis (SSCA), denaturing gradient gel electrophoresis (DGGE),heteroduplex analysis (HET), chemical cleavage analysis (CCM), catalyticnucleic acid cleavage or a combination thereof (see, for example,Lemieux, 2000; Langridge et al., 2001). The invention also includes theuse of molecular marker techniques to detect polymorphisms linked toalleles of (for example) a FAD2, FATB or FAD6 gene which confer thedesired phenotype. Such methods include the detection or analysis ofrestriction fragment length polymorphisms (RFLP), RAPD, amplifiedfragment length polymorphisms (AFLP) and microsatellite (simple sequencerepeat, SSR) polymorphisms. The closely linked markers can be obtainedreadily by methods well known in the art, such as Bulked SegregantAnalysis, as reviewed by Langridge et al. (2001).

The “polymerase chain reaction” (“PCR”) is a reaction in which replicatecopies are made of a target polynucleotide using a “pair of primers” or“set of primers” consisting of “upstream” and a “downstream” primer, anda catalyst of polymerization, such as a DNA polymerase, and typically athermally-stable polymerase enzyme. Methods for PCR are known in theart, and are taught, for example, in “PCR” (Ed. M. J. McPherson and S. GMoller (2000) BIOS Scientific Publishers Ltd, Oxford). PCR can beperformed on cDNA obtained from reverse transcribing mRNA isolated fromplant cells. However, it will generally be easier if PCR is performed ongenomic DNA isolated from a plant.

A primer is an oligonucleotide sequence that is capable of hybridisingin a sequence specific fashion to the target sequence and being extendedduring the PCR. Amplicons or PCR products or PCR fragments oramplification products are extension products that comprise the primerand the newly synthesized copies of the target sequences. Multiplex PCRsystems contain multiple sets of primers that result in simultaneousproduction of more than one amplicon. Primers may be perfectly matchedto the target sequence or they may contain internal mismatched basesthat can result in the introduction of restriction enzyme or catalyticnucleic acid recognition/cleavage sites in specific target sequences.Primers may also contain additional sequences and/or contain modified orlabelled nucleotides to facilitate capture or detection of amplicons.Repeated cycles of heat denaturation of the DNA, annealing of primers totheir complementary sequences and extension of the annealed primers withpolymerase result in exponential amplification of the target sequence.The terms target or target sequence or template refer to nucleic acidsequences which are amplified.

Methods for direct sequencing of nucleotide sequences are well known tothose skilled in the art and can be found for example in Ausubel et al.(supra) and Sambrook et al. (supra). Sequencing can be carried out byany suitable method, for example, dideoxy sequencing, chemicalsequencing or variations thereof. Direct sequencing has the advantage ofdetermining variation in any base pair of a particular sequence.

Hybridization based detection systems include, but are not limited to,the TaqMan assay and molecular beacon assay (U.S. Pat. No. 5,925,517).The TaqMan assay (U.S. Pat. No. 5,962,233) uses allele specific (ASO)probes with a donor dye on one end and an acceptor dye on the other endsuch that the dye pair interact via fluorescence resonance energytransfer (FRET).

In one embodiment, the method described in Example 7 is used inselection and breeding programs to identify and select safflower plantswith the ol mutation. For instance, the method comprises performing anamplification reaction on genomic DNA obtained from the plant using oneor both of the following sets of primers;

i) 5′-ATAAGGCTGTGTTCACGGGTTT-3′ (SEQ ID NO: 140) and5′-GCTCAGTTGGGGATACAAGGAT-3′, (SEQ ID NO: 141) andii) 5′-AGTTATGGTTCGATGATCGACG-3′ (SEQ ID NO: 142) and5′-TTGCTATACATATTGAAGGCACT -3. (SEQ ID NO: 143)Polypeptides

The terms “polypeptide” and “protein” are generally usedinterchangeably. A polypeptide or class of polypeptides may be definedby the extent of identity (% identity) of its amino acid sequence to areference amino acid sequence, or by having a greater % identity to onereference amino acid sequence than to another. The % identity of apolypeptide to a reference amino acid sequence is typically determinedby GAP analysis (Needleman and Wunsch, 1970; GCG program) withparameters of a gap creation penalty=5, and a gap extension penalty=0.3.The query sequence is at least 100 amino acids in length and the GAPanalysis aligns the two sequences over a region of at least 100 aminoacids. Even more preferably, the query sequence is at least 250 aminoacids in length and the GAP analysis aligns the two sequences over aregion of at least 250 amino acids. Even more preferably, the GAPanalysis aligns two sequences over their entire length. The polypeptideor class of polypeptides may have the same enzymatic activity as, or adifferent activity than, or lack the activity of, the referencepolypeptide. Preferably, the polypeptide has an enzymatic activity of atleast 10% of the activity of the reference polypeptide.

As used herein a “biologically active fragment” is a portion of apolypeptide of the invention which maintains a defined activity of afull-length reference polypeptide for example, Δ12 desaturase,palmitoyl-ACP thioesterase or Δ6 desaturase activity. Biologicallyactive fragments as used herein exclude the full-length polypeptide.Biologically active fragments can be any size portion as long as theymaintain the defined activity. Preferably, the biologically activefragment maintains at least 10% of the activity of the full lengthpolypeptide.

With regard to a defined polypeptide or enzyme, it will be appreciatedthat % identity figures higher than those provided herein will encompasspreferred embodiments. Thus, where applicable, in light of the minimum %identity figures, it is preferred that the polypeptide/enzyme comprisesan amino acid sequence which is at least 50%, more preferably at least60%, more preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, more preferably at least 93%, morepreferably at least 94%, more preferably at least 95%, more preferablyat least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99%, more preferably at least 99.1%, morepreferably at least 99.2%, more preferably at least 99.3%, morepreferably at least 99.4%, more preferably at least 99.5%, morepreferably at least 99.6%, more preferably at least 99.7%, morepreferably at least 99.8%, and even more preferably at least 99.9%identical to the relevant nominated SEQ ID NO.

Amino acid sequence mutants of the polypeptides defined herein can beprepared by introducing appropriate nucleotide changes into a nucleicacid defined herein, or by in vitro synthesis of the desiredpolypeptide. Such mutants include for example, deletions, insertions, orsubstitutions of residues within the amino acid sequence. A combinationof deletions, insertions and substitutions can be made to arrive at thefinal construct, provided that the final polypeptide product possessesthe desired characteristics.

Mutant (altered) polypeptides can be prepared using any technique knownin the art, for example, using directed evolution or rationale designstrategies (see below). Products derived from mutated/altered DNA canreadily be screened using techniques described herein to determine ifthey possess, or lack, Δ12 desaturase, palmitoyl-ACP thioesterase or ω6Δ6 desaturase activity.

In designing amino acid sequence mutants, the location of the mutationsite and the nature of the mutation will depend on characteristic(s) tobe modified. The sites for mutation can be modified individually or inseries for example, by (1) substituting first with conservative aminoacid choices and then with more radical selections depending upon theresults achieved, (2) deleting the target residue, or (3) insertingother residues adjacent to the located site. As the skilled person willappreciate, the use of non-conservative substitutions can be used whenproducing a mutant with reduced enzymatic activity.

Amino acid sequence deletions generally range from about 1 to 15residues, more preferably about 1 to 10 residues and typically about 1to 5 contiguous residues, but may be much longer particularly when themutant is designed to reduce enzymatic activity.

Substitution mutants have at least one amino acid residue in thepolypeptide removed and a different residue inserted in its place. Thesites of greatest interest for substitutional mutagenesis to inactivatean enzyme include sites identified as the active site(s). Other sites ofinterest are those in which particular residues obtained from variousstrains or species are identical. These positions may be important forbiological activity. Conservative substitutions are shown in Table 1,while non-conservative substitutions are substitutions which are notconservative substitutions. In one embodiment, a polypeptide of theinvention is a Δ12 desaturase and which comprises amino acids having asequence as provided in any one of SEQ ID NOs: 27 to 34, 36 or 37, abiologically active fragment thereof, or an amino acid sequence which isat least 40% identical to any one or more of SEQ ID NOs: NOs: 27 to 34,36 or 37.

In another embodiment, a polypeptide of the invention is oleate Δ12desaturase present in the seed (oilseed) of an oilseed plant whichcomprises amino acids having a sequence as provided in any one of SEQ IDNOs: 27, 28 or 36, a biologically active fragment thereof, or an aminoacid sequence which is at least 40% identical to any one or more of SEQID NOs: 27, 28 or 36.

In another embodiment, a polypeptide of the invention is aΔ12-acetylenase which comprises amino acids having a sequence asprovided in SEQ ID NO:37, a biologically active fragment thereof, or anamino acid sequence which is at least 40% identical to SEQ ID NO:37.

In another embodiment, a polypeptide of the invention is a palmitoleateΔ12 desaturase which comprises amino acids having a sequence as providedin SEQ ID NO:35, a biologically active fragment thereof, or an aminoacid sequence which is at least 40% identical to SEQ ID NO:35.

In another embodiment, a polypeptide of the invention is a palmitoyl-ACPthioesterase (FATB) which comprises amino acids having a sequence asprovided in any one of SEQ ID NOs: 44 or 45, a biologically activefragment thereof, or an amino acid sequence which is at least 40%identical to any one or more of SEQ ID NOs: 44 or 45.

TABLE 1 Conservative substitutions. Original Residue ExemplarySubstitutions Ala (A) val; leu; ile; gly Arg (R) lys Asn (N) gln; hisAsp (D) glu Cys (C) ser Gln (Q) asn; his Glu (E) asp Gly (G) pro, alaHis (H) asn; gln Ile (I) leu; val; ala Leu (L) ile; val; met; ala; pheLys (K) arg Met (M) leu; phe Phe (F) leu; val; ala Pro (P) gly Ser (S)thr Thr (T) ser Trp (W) tyr Tyr (Y) trp; phe Val (V) ile; leu; met; phe,ala

In another embodiment, a polypeptide of the invention is a palmitoyl-ACPthioesterase (FATB) present in the seed of an oilseed plant whichcomprises amino acids having a sequence as provided in SEQ ID NO:45, abiologically active fragment thereof, or an amino acid sequence which isat least 40% identical to SEQ ID NO:45.

In another embodiment, a polypeptide of the invention is a 46 desaturasewhich comprises amino acids having a sequence as provided in SEQ IDNO:48, a biologically active fragment thereof, or an amino acid sequencewhich is at least 40% identical to SEQ ID NO:48.

Preferred features of the enzymes of the invention are provided in theExamples section, in particular Example 2 in relation to safflowerFAD2's.

Polypeptides as described herein may be expressed as a fusion to atleast one other polypeptide. In a preferred embodiment, the at least oneother polypeptide is selected from the group consisting of: apolypeptide that enhances the stability of the fusion protein, and apolypeptide that assists in the purification of the fusion protein.

Production of Lipids and/or Oils High in Oleic Acid

Techniques that are routinely practiced in the art can be used toextract, process, purify and analyze the lipids produced by the plants,in particular the seeds, of the instant invention. Such techniques aredescribed and explained throughout the literature in sources such as,Fereidoon Shahidi, Current Protocols in Food Analytical Chemistry, JohnWiley & Sons, Inc. (2001) D1.1.1-D1.1.11, and Perez-Vich et al. (1998).

Production of Seedoil

Typically, plant seeds are cooked, pressed, and/or extracted to producecrude seedoil, which is then degummed, refined, bleached, anddeodorized. Generally, techniques for crushing seed are known in theart. For example, oilseeds can be tempered by spraying them with waterto raise the moisture content to, for example, 8.5%, and flaked using asmooth roller with a gap setting of 0.23 to 0.27 mm. Depending on thetype of seed, water may not be added prior to crushing. Application ofheat deactivates enzymes, facilitates further cell rupturing, coalescesthe lipid droplets, and agglomerates protein particles, all of whichfacilitate the extraction process.

In an embodiment, the majority of the seedoil is released by passagethrough a screw press. Cakes expelled from the screw press are thensolvent extracted for example, with hexane, using a heat traced column.Alternatively, crude seedoil produced by the pressing operation can bepassed through a settling tank with a slotted wire drainage top toremove the solids that are expressed with the seedoil during thepressing operation. The solid residue from the pressing and extraction,after removal of the hexane, is the seedmeal, which is typically used asanimal feed. The clarified seedoil can be passed through a plate andframe filter to remove any remaining fine solid particles. If desired,the seedoil recovered from the extraction process can be combined withthe clarified seedoil to produce a blended crude seedoil.

Once the solvent is stripped from the crude seedoil, the pressed andextracted portions are combined and subjected to normal lipid processingprocedures such as, for example, degumming, caustic refining, bleaching,and deodorization. Some or all steps may be omitted, depending on thenature of the product path, e.g. for feed grade oil, limited treatmentmay be needed whereas for oleochemical applications, more purificationsteps are required.

Degumming

Degumming is an early step in the refining of oils and its primarypurpose is the removal of most of the phospholipids from the oil, whichmay be present as approximately 1-2% of the total extracted lipid.Addition of ˜2% of water, typically containing phosphoric acid, at70-80° C. to the crude oil results in the separation of most of thephospholipids accompanied by trace metals and pigments. The insolublematerial that is removed is mainly a mixture of phospholipids andtriacylglycerols and is also known as lecithin. Degumming can beperformed by addition of concentrated phosphoric acid to the crudeseedoil to convert non-hydratable phosphatides to a hydratable form, andto chelate minor metals that are present. Gum is separated from theseedoil by centrifugation.

Alkali Refining

Alkali refining is one of the refining processes for treating crude oil,sometimes also referred to as neutralization. It usually followsdegumming and precedes bleaching. Following degumming, the seedoil cantreated by the addition of a sufficient amount of an alkali solution totitrate all of the fatty acids and phosphoric acids, and removing thesoaps thus formed. Suitable alkaline materials include sodium hydroxide,potassium hydroxide, sodium carbonate, lithium hydroxide, calciumhydroxide, calcium carbonate and ammonium hydroxide. This process istypically carried out at room temperature and removes the free fattyacid fraction. Soap is removed by centrifugation or by extraction into asolvent for the soap, and the neutralised oil is washed with water. Ifrequired, any excess alkali in the oil may be neutralized with asuitable acid such as hydrochloric acid or sulphuric acid.

Bleaching

Bleaching is a refining process in which oils are heated at 90-120° C.for 10-30 minutes in the presence of a bleaching earth (0.2-2.0%) and inthe absence of oxygen by operating with nitrogen or steam or in avacuum. This step in oil processing is designed to remove unwantedpigments (carotenoids, chlorophyll, gossypol etc), and the process alsoremoves oxidation products, trace metals, sulphur compounds and tracesof soap.

Deodorization

Deodorization is a treatment of oils and fats at a high temperature(200-260° C.) and low pressure (0.1-1 mm Hg). This is typically achievedby introducing steam into the seedoil at a rate of about 0.1ml/minute/100 ml of seedoil. After about 30 minutes of sparging, theseedoil is allowed to cool under vacuum. The seedoil is typicallytransferred to a glass container and flushed with argon before beingstored under refrigeration. This treatment improves the colour of theseedoil and removes a majority of the volatile substances or odorouscompounds including any remaining free fatty acids, monoacylglycerolsand oxidation products.

Winterisation

Winterization is a process sometimes used in commercial production ofoils for the separation of oils and fats into solid (stearin) and liquid(olein) fractions by crystallization at sub-ambient temperatures. It wasapplied originally to cottonseed oil to produce a solid-free product. Itis typically used to decrease the saturated fatty acid content of oils.

Transesterification

Transesterification is a process that exchanges the fatty acids withinand between TAGs, initially by releasing fatty acids from the TAGseither as free fatty acids or as fatty acid esters, usually fatty acidethyl esters. When combined with a fractionation process,transesterification can be used to modify the fatty acid composition oflipids (Marangoni et al., 1995). Transesterification can use eitherchemical or enzymatic means, the latter using lipases which may beposition-specific (sn-⅓ or sn-2 specific) for the fatty acid on the TAG,or having a preference for some fatty acids over others (Speranza et al,2012). The fatty acid fractionation to increase the concentration ofLC-PUFA in an oil can be achieved by any of the methods known in theart, such as, for example, freezing crystallization, complex formationusing urea, molecular distillation, supercritical fluid extraction andsilver ion complexing. Complex formation with urea is a preferred methodfor its simplicity and efficiency in reducing the level of saturated andmonounsaturated fatty acids in the oil (Gamez et al., 2003). Initially,the TAGs of the oil are split into their constituent fatty acids, oftenin the form of fatty acid esters, by hydrolysis or by lipases and thesefree fatty acids or fatty acid esters are then mixed with an ethanolicsolution of urea for complex formation. The saturated andmonounsaturated fatty acids easily complex with urea and crystallize outon cooling and may subsequently be removed by filtration. The non-ureacomplexed fraction is thereby enriched with LC-PUFA.

Hydrogenation

Hydrogenation of fatty acids involves treatment with hydrogen, typicallyin the presence of a catalyst. Non-catalytic hydrogenation takes placeonly at very high temperatures.

Hydrogenation is commonly used in the processing of plant oils.Hydrogenation converts unsaturated fatty acids to saturated fatty acids,and in some cases, trans fats. Hydrogenation results in the conversionof liquid plant oils to solid or semi-solid fats, such as those presentin margarine. Changing the degree of saturation of the fat changes someimportant physical properties such as the melting range, which is whyliquid oils become semi-solid. Solid or semi-solid fats are preferredfor baking because the way the fat mixes with flour produces a moredesirable texture in the baked product. Because partially hydrogenatedvegetable oils are cheaper than animal source fats, are available in awide range of consistencies, and have other desirable characteristics(e.g., increased oxidative stability/longer shelf life), they are thepredominant fats used as shortening in most commercial baked goods.

In an embodiment, the lipid/oil of the invention has not beenhydrogenated. An indication that a lipid or oil has not beenhydrogenated is the absence of any trans fatty acids in its TAG.

Uses of Lipids

The lipids such as the seedoil, preferably the safflower seedoil,produced by the methods described herein have a variety of uses. In someembodiments, the lipids are used as food oils. In other embodiments, thelipids are refined and used as lubricants or for other industrial usessuch as the synthesis of plastics. It may be used in the manufacture ofcosmetics, soaps, fabric softeners, electrical insulation or detergents.It may be used to produce agricultural chemicals such as surfactants oremulsifiers. In some embodiments, the lipids are refined to producebiodiesel. The oil of the invention may advantageously be used in paintsor varnishes since the absence of linolenic acid means it does notdiscolour easily.

An industrial product produced using a method of the invention may be ahydrocarbon product such as fatty acid esters, preferably fatty acidmethyl esters and/or a fatty acid ethyl esters, an alkane such asmethane, ethane or a longer-chain alkane, a mixture of longer chainalkanes, an alkene, a biofuel, carbon monoxide and/or hydrogen gas, abioalcohol such as ethanol, propanol, or butanol, biochar, or acombination of carbon monoxide, hydrogen and biochar. The industrialproduct may be a mixture of any of these components, such as a mixtureof alkanes, or alkanes and alkenes, preferably a mixture which ispredominantly (>50%) C4-C8 alkanes, or predominantly C6 to C10 alkanes,or predominantly C6 to C8 alkanes. The industrial product is not carbondioxide and not water, although these molecules may be produced incombination with the industrial product. The industrial product may be agas at atmospheric pressure/room temperature, or preferably, a liquid,or a solid such as biochar, or the process may produce a combination ofa gas component, a liquid component and a solid component such as carbonmonoxide, hydrogen gas, alkanes and biochar, which may subsequently beseparated. In an embodiment, the hydrocarbon product is predominantlyfatty acid methyl esters. In an alternative embodiment, the hydrocarbonproduct is a product other than fatty acid methyl esters.

Heat may be applied in the process, such as by pyrolysis, combustion,gasification, or together with enzymatic digestion (including anaerobicdigestion, composting, fermentation). Lower temperature gasificationtakes place at, for example, between about 700° C. to about 1000° C.Higher temperature gasification takes place at, for example, betweenabout 1200° C. to about 1600° C. Lower temperature pyrolysis (slowerpyrolysis), takes place at about 400° C., whereas higher temperaturepyrolysis takes place at about 500° C. Mesophilic digestion takes placebetween about 20° C. and about 40° C. Thermophilic digestion takes placefrom about 50° C. to about 65° C.

Chemical means include, but are not limited to, catalytic cracking,anaerobic digestion, fermentation, composting and transesterification.In an embodiment, a chemical means uses a catalyst or mixture ofcatalysts, which may be applied together with heat. The process may usea homogeneous catalyst, a heterogeneous catalyst and/or an enzymaticcatalyst. In an embodiment, the catalyst is a transition metal catalyst,a molecular sieve type catalyst, an activated alumina catalyst or sodiumcarbonate as a catalyst. Catalysts include acid catalysts such assulphuric acid, or alkali catalysts such as potassium or sodiumhydroxide or other hydroxides. The chemical means may comprisetransesterification of fatty acids in the lipid, which process may use ahomogeneous catalyst, a heterogeneous catalyst and/or an enzymaticcatalyst. The conversion may comprise pyrolysis, which applies heat andmay apply chemical means, and may use a transition metal catalyst, amolecular sieve type catalyst, an activated alumina catalyst and/orsodium carbonate as a catalyst.

Enzymatic means include, but are not limited to, digestion bymicroorganisms in, for example, anaerobic digestion, fermentation orcomposting, or by recombinant enzymatic proteins.

Biofuel

As used herein the term “biofuel” includes biodiesel and bioalcohol.Biodiesel can be made from oils derived from plants, algae and fungi.Bioalcohol is produced from the fermentation of sugar. This sugar can beextracted directly from plants (e.g., sugarcane), derived from plantstarch (e.g., maize or wheat) or made from cellulose (e.g., wood, leavesor stems).

Biofuels currently cost more to produce than petroleum fuels. Inaddition to processing costs, biofuel crops require planting,fertilising, pesticide and herbicide applications, harvesting andtransportation. Plants, algae and fungi of the present invention mayreduce production costs of biofuel.

General methods for the production of biofuel can be found in, forexample, Maher and Bressler (2006), Maher and Bressler (2007), Greenwellet al. (2011), Karmakar et al. (2010), Alonso et al. (2010), Lee andMohamed (2010), Liu et al. (2010), Gong and Jiang (2011), Endalew et al.(2011) and Semwal et al. (2011).

Biodiesel

The production of biodiesel, or alkyl esters, is well known. There arethree basic routes to ester production from lipids: 1) Base catalysedtransesterification of the lipid with alcohol; 2) Direct acid catalysedesterification of the lipid with methanol; and 3) Conversion of thelipid to fatty acids, and then to alkyl esters with acid catalysis.

Any method for preparing fatty acid alkyl esters and glyceryl ethers (inwhich one, two or three of the hydroxy groups on glycerol areetherified) can be used. For example, fatty acids can be prepared, forexample, by hydrolyzing or saponifying triglycerides with acid or basecatalysts, respectively, or using an enzyme such as a lipase or anesterase. Fatty acid alkyl esters can be prepared by reacting a fattyacid with an alcohol in the presence of an acid catalyst. Fatty acidalkyl esters can also be prepared by reacting a triglyceride with analcohol in the presence of an acid or base catalyst. Glycerol ethers canbe prepared, for example, by reacting glycerol with an alkyl halide inthe presence of base, or with an olefin or alcohol in the presence of anacid catalyst.

In some preferred embodiments, the lipids are transesterified to producemethyl esters and glycerol. In some preferred embodiments, the lipidsare reacted with an alcohol (such as methanol or ethanol) in thepresence of a catalyst (for example, potassium or sodium hydroxide) toproduce alkyl esters. The alkyl esters can be used for biodiesel orblended with petroleum based fuels.

The alkyl esters can be directly blended with diesel fuel, or washedwith water or other aqueous solutions to remove various impurities,including the catalysts, before blending. It is possible to neutralizeacid catalysts with base. However, this process produces salt. To avoidengine corrosion, it is preferable to minimize the salt concentration inthe fuel additive composition. Salts can be substantially removed fromthe composition, for example, by washing the composition with water.

In another embodiment, the composition is dried after it is washed, forexample, by passing the composition through a drying agent such ascalcium sulfate.

In yet another embodiment, a neutral fuel additive is obtained withoutproducing salts or using a washing step, by using a polymeric acid, suchas Dowex 50™, which is a resin that contains sulfonic acid groups. Thecatalyst is easily removed by filtration after the esterification andetherification reactions are complete.

Plant Triacylglycerols as a Biofuel Source

Use of plant triacylglycerols for the production of biofuel is reviewedin Durrett et al. (2008). Briefly, plant oils are primarily composed ofvarious triacylglycerols (TAGs), molecules that consist of three fattyacid chains (usually 18 or 16 carbons long) esterified to glycerol. Thefatty acyl chains are chemically similar to the aliphatic hydrocarbonsthat make up the bulk of the molecules found in petrol and diesel. Thehydrocarbons in petrol contain between 5 and 12 carbon atoms permolecule, and this volatile fuel is mixed with air and ignited with aspark in a conventional engine. In contrast, diesel fuel componentstypically have 10-15 carbon atoms per molecule and are ignited by thevery high compression obtained in a diesel engine. However, most plantTAGs have a viscosity range that is much higher than that ofconventional diesel: 17.3-32.9 mm² s⁻¹ compared to 1.9-4.1 mm² s⁻¹,respectively (ASTM D975; Knothe and Steidley, 2005). This higherviscosity results in poor fuel atomization in modern diesel engines,leading to problems derived from incomplete combustion such as carbondeposition and coking (Ryan et al., 1984). To overcome this problem,TAGs are converted to less viscous fatty acid esters by esterificationwith a primary alcohol, most commonly methanol. The resulting fuel iscommonly referred to as biodiesel and has a dynamic viscosity range from1.9 to 6.0 mm²s⁻¹ (ASTM D6751). The fatty acid methyl esters (FAMEs)found in biodiesel have a high energy density as reflected by their highheat of combustion, which is similar, if not greater, than that ofconventional diesel (Knothe, 2005). Similarly, the cetane number (ameasure of diesel ignition quality) of the FAMEs found in biodieselexceeds that of conventional diesel (Knothe, 2005).

Plant oils are mostly composed of five common fatty acids, namelypalmitate (16:0), stearate (18:0), oleate (18:1), linoleate (18:2) andlinolenate (18:3), although, depending on the particular species, longeror shorter fatty acids may also be major constituents. These fatty acidsdiffer from each other in terms of acyl chain length and number ofdouble bonds, leading to different physical properties. Consequently,the fuel properties of biodiesel derived from a mixture of fatty acidsare dependent on that composition. Altering the fatty acid profile cantherefore improve fuel properties of biodiesel such as cold-temperatureflow characteristics, oxidative stability and NOx emissions. Alteringthe fatty acid composition of TAGs may reduce the viscosity of the plantoils, eliminating the need for chemical modification, thus improving thecost-effectiveness of biofuels.

Feedstuffs

The lipid/oil of the invention has advantages in food applicationsbecause of its very high oleic acid content and the low levels oflinoleic acid (<3.2%) and saturated fatty acids such as palmitic acid,and the essentially zero level of linolenic acid. This provides the oilwith a high oxidative stability, producing less rancidity and making itideal for food applications where heating is required, such as in fryingapplications, for example for French fries. The oil has a high OSI(oxidative stability index) which is measured as the length of time anoil may be held at 110° C., such as greater than 20 or hours, preferablygreater than 30 hours or greater than 50 hours. The low levels ofsaturated fatty acids relative to other vegetable oils provides forhealth benefits since saturated fatty acids have been associated withdeleterious effects on health. The oils also have essentially zero transfatty acid content which is desirable in some markets as trans fattyacids have also been associated with negative effects on heart health orraising LDL cholesterol. Moreover, due to its very low level ofpolyunsaturated fatty acids, the oil does not require hydrogenation tolower the levels of PUFA—such hydrogenation produces trans fatty acids.The oils are also advantageous for reducing the incidence or severity ofobesity and diabetes. They are also desirable for food applications inthat they contain only naturally occurring fatty acids (Scarth and Tang,2006).

For purposes of the present invention, “feedstuffs” include any food orpreparation for human or animal consumption (including for enteraland/or parenteral consumption) which when taken into the body: (1) serveto nourish or build up tissues or supply energy, and/or (2) maintain,restore or support adequate nutritional status or metabolic function.Feedstuffs of the invention include nutritional compositions for babiesand/or young children.

Feedstuffs of the invention comprise for example, a cell of theinvention, a plant of the invention, the plant part of the invention,the seed of the invention, an extract of the invention, the product of amethod of the invention, or a composition along with a suitablecarrier(s). The term “carrier” is used in its broadest sense toencompass any component which may or may not have nutritional value. Asthe person skilled in the art will appreciate, the carrier must besuitable for use (or used in a sufficiently low concentration) in afeedstuff, such that it does not have deleterious effect on an organismwhich consumes the feedstuff.

The feedstuff of the present invention comprises a lipid produceddirectly or indirectly by use of the methods, cells or organismsdisclosed herein. The composition may either be in a solid or liquidform. Additionally, the composition may include edible macronutrients,vitamins, and/or minerals in amounts desired for a particular use. Theamounts of these or other ingredients will vary depending on whether thecomposition is intended for use with normal individuals or for use withindividuals having specialized needs such as individuals suffering frommetabolic disorders and the like.

The foods may be produced by mixing the oil with one or more otheringredients so that the food comprises the oil, or mixed with one ormore other ingredients to make a food additive such as salad dressing ormayonnaise. The food or food additive may comprise 1%-10% or more of theoil by weight. The oil may be blended with other vegetable oils toprovide for optimal composition or with solid fats or with palm oil toprovide semisolid shortening. Foods or food additives produced from theoil include salad dressing, mayonnaise, margarine, bread, cakes,biscuits (cookies), croissants, baked goods, pancakes or pancake mixes,custards, frozen desserts, non-dairy foods.

Examples of suitable carriers with nutritional value include, but arenot limited to, macronutrients such as edible fats, carbohydrates andproteins. Examples of such edible fats include, but are not limited to,coconut oil, borage oil, fungal oil, black current oil, soy oil, andmono- and di-glycerides. Examples of such carbohydrates include, but arenot limited to, glucose, edible lactose, and hydrolyzed starch.Additionally, examples of proteins which may be utilized in thenutritional composition of the invention include, but are not limitedto, soy proteins, electrodialysed whey, electrodialysed skim milk, milkwhey, or the hydrolysates of these proteins.

With respect to vitamins and minerals, the following may be added to thefeedstuff compositions of the present invention, calcium, phosphorus,potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc,selenium, iodine, and vitamins A, E, D, C, and the B complex. Other suchvitamins and minerals may also be added.

The components utilized in the feedstuff compositions of the presentinvention can be of semi-purified or purified origin. By semi-purifiedor purified is meant a material which has been prepared by purificationof a natural material.

A feedstuff composition of the present invention may also be added tofood even when supplementation of the diet is not required. For example,the composition may be added to food of any type, including, but notlimited to, margarine, modified butter, cheeses, milk, yogurt,chocolate, candy, snacks, salad oils, cooking oils, cooking fats, meats,fish and beverages.

Additionally, lipid produced in accordance with the present invention orhost cells transformed to contain and express the subject genes may alsobe used as animal food supplements to alter an animal's tissue or milkfatty acid composition or fatty acod composition of eggs, to one moredesirable for human or animal consumption, or for animal health andwellbeing. Examples of such animals include sheep, cattle, horses,poultry, pets such as dogs and cats and the like.

Furthermore, feedstuffs of the invention can be used in aquaculture toincrease the levels of fatty acids in fish for human or animalconsumption.

Preferred feedstuffs of the invention are the plants, seed and otherplant parts such as leaves, fruits and stems which may be used directlyas food or feed for humans or other animals. For example, animals maygraze directly on such plants grown in the field, or be fed moremeasured amounts in controlled feeding.

Compositions

The present invention also encompasses compositions, particularlypharmaceutical compositions, comprising one or more lipids or oilsproduced using the methods of the invention.

A pharmaceutical composition may comprise one or more of the lipids, incombination with a standard, well-known, non-toxicpharmaceutically-acceptable carrier, adjuvant or vehicle such asphosphate-buffered saline, water, ethanol, polyols, vegetable oils, awetting agent, or an emulsion such as a water/oil emulsion. Thecomposition may be in either a liquid or solid form. For example, thecomposition may be in the form of a tablet, capsule, ingestible liquid,powder, topical ointment or cream. Proper fluidity can be maintained forexample, by the maintenance of the required particle size in the case ofdispersions and by the use of surfactants. It may also be desirable toinclude isotonic agents for example, sugars, sodium chloride, and thelike. Besides such inert diluents, the composition can also includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening agents, flavoring agents and perfuming agents.

Suspensions, in addition to the active compounds, may comprisesuspending agents such as ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth,or mixtures of these substances.

Solid dosage forms such as tablets and capsules can be prepared usingtechniques well known in the art. For example, lipid produced inaccordance with the present invention can be tableted with conventionaltablet bases such as lactose, sucrose, and cornstarch in combinationwith binders such as acacia, cornstarch or gelatin, disintegratingagents such as potato starch or alginic acid, and a lubricant such asstearic acid or magnesium stearate. Capsules can be prepared byincorporating these excipients into a gelatin capsule along withantioxidants and the relevant lipid(s).

For intravenous administration, the lipids produced in accordance withthe present invention or derivatives thereof may be incorporated intocommercial formulations.

A typical dosage of a particular fatty acid is from 0.1 mg to 20 g,taken from one to five times per day (up to 100 g daily) and ispreferably in the range of from about 10 mg to about 1, 2, 5, or 10 gdaily (taken in one or multiple doses). As known in the art, a minimumof about 300 mg/day of fatty acid is desirable. However, it will beappreciated that any amount of fatty acid will be beneficial to thesubject.

Possible routes of administration of the pharmaceutical compositions ofthe present invention include for example, enteral and parenteral. Forexample, a liquid preparation may be administered orally. Additionally,a homogenous mixture can be completely dispersed in water, admixed understerile conditions with physiologically acceptable diluents,preservatives, buffers or propellants to form a spray or inhalant.

The dosage of the composition to be administered to the subject may bedetermined by one of ordinary skill in the art and depends upon variousfactors such as weight, age, overall health, past history, immunestatus, etc., of the subject.

Additionally, the compositions of the present invention may be utilizedfor cosmetic purposes. The compositions may be added to pre-existingcosmetic compositions, such that a mixture is formed, or a fatty acidproduced according to the invention may be used as the sole “active”ingredient in a cosmetic composition.

EXAMPLES Example 1. General Materials and Methods

Plant Materials and Growth Conditions

Safflower plants (Carthamus tinctorius) genotypes SU, S-317, 5-517,LeSaf496, CW99-OL, and Ciano-OL were grown from seed in the glasshousein a perlite and sandy loam potting mix under a day/night cycle of 16hrs (25° C.)/8 hrs (22° C.). The wild type variety SU, which is a highlinoleic variety, was obtained from Heffeman Seeds in NSW. Seeds of PI603208 (LeSaf496, ATC 120562) and CW 99-OL (ATC 120561) were obtainedfrom the Australian Temperate Field Crops Collection.

Plant tissues for DNA and RNA extraction including leaves, roots,cotyledons and hypocotyls were harvested from safflower seedlings 10days post-germination. Flowering heads were obtained at the first day offlower opening and developing embryos were harvested at threedevelopmental stages at 7 (early), 15 (middle) and 20 (late) days postanthesis (DPA). Samples were immediately chilled in liquid nitrogen andstored at −80° C. until DNA and RNA extraction was carried out.

Safflower florets are tubular and largely self-pollinating withgenerally less than 10% outcrossing (Knowles 1969). Insects, but notwind, can increase levels of out crossing in the field. The unpollinatedstigma may remain receptive for several days. Each capitula (safflowerhead) contains about 15-30 achenes. Seed mass of the developing seed inthe plant increases rapidly during the first 15 days after flowering.Oil content increases 5- to 10-fold during the period of 10-15 DAP andreaches a maximum level at about 28 DPA (Hill and Knowles 1968).Safflower seed and plants are physiologically mature about 5 weeks afterflowering and the seed ready to harvest when most of the leaves haveturned brown and only a tint of green remains on the bracts of thelatest flowering heads. Seeds were readily harvested by rubbing theheads by hand.

Lipid Analysis

Isolation of Lipid Samples from Single Seeds for Rapid Fatty AcidComposition Analysis

After being harvested at plant maturity, safflower seeds were dried bystoring the seeds for 3 days at 37° C. and subsequently at roomtemperature if not analysed right away. Single seeds or pooled seedswere crushed between small filter papers and the exuded seedoil samplesthat soaked into the papers analysed for fatty acid composition by GCmethods as described below.

Total Lipid Isolation from Half Cotyledons Post Germination

For screening purposes, for example for progeny seeds from transgenicplants, safflower seeds were germinated on a wet filter paper in a petridish for 1 day. A cotyledon was carefully removed from each germinatedseed for lipid analysis. The remainder of each seedling was transferredto soil and the resultant plants grown to maturity followed byharvesting of seeds to maintain the transgenic line.

Extraction of Oil from Seeds Using Soxhlet Apparatus

For quantitative extraction of seedoil, harvested safflower seeds weredried in an oven at 105° C. overnight and then ground in a Puck Mill for1 min. The ground seed material (˜250 grams) was collected into apre-weighed thimble and weighed prior to oil extraction. After adding alayer of cotton wool on top of the meal, the oil was extracted in aSoxhlet Extraction apparatus with solvent (Petroleum Spirit 40-60 C),initially at 70-80° C. The mixture was then refluxed overnight with thesolvent syphoning to the extraction flask every 15-20 min. Thedissolved, extracted oil was recovered by evaporating off the solventusing a rotary evaporator under vacuum. The weight of the extracted oilwas measured and the oil content was determined. To determine the fattyacid composition of the extracted oil, small aliquots were diluted inchloroform and analysed by gas chromatography.

Total Lipid Isolation from Leaf Material

Leaf tissue samples were freeze-dried, weighed and total lipidsextracted from samples of approximately 10 mg dry weight as described byBligh and Dyer (1959).

Fractionation of Lipids

When required, TAG fractions were separated from other lipid componentsusing a 2-phase thin-layer chromatography (TLC) system on pre-coatedsilica gel plates (Silica gel 60, Merck). An extracted lipid sampleequivalent to 10 mg dry weight of leaf tissue was chromatographed in afirst phase with hexane/diethyl ether (98/2 v/v) to remove non-polarwaxes and then in a second phase using hexane/diethyl ether/acetic acid(70/30/1 v/v/v). When required, polar lipids were separated fromnon-polar lipids in lipid samples extracted from an equivalent of 5 mgdry weight of leaves using two-dimensional TLC (Silica gel 60, Merck),using chloroform/methanol/water (65/25/4 v/v/v) for the first directionand chloroform/methanol/NH₄OH/ethylpropylamine (130/70/10/1 v/v/v/v) forthe second direction. The lipid spots, and appropriate standards run onthe same TLC plates, were visualized by brief exposure to iodine vapour,collected into vials and transmethylated to produce FAME for GC analysisas follows.

Fatty Acid Methyl Esters (FAME) Preparation and Gas Chromatography (GC)Analysis

For fatty acid composition analysis by GC, extracted lipid samplesprepared as described above were transferred to a glass tube andtransmethylated in 2 mL of 1 M HCl in methanol (Supelco) at 80° C. for 3hours. After cooling to room temperature, 1.3 mL 0.9% NaCl and 800 μLhexane were added to each tube and FAMEs were extracted into the hexanephase. To determine the fatty acid composition, the FAMEs were separatedby gas-chromatography (GC) using an Agilent Technologies 7890A gaschromatograph (Palo Alto, Calif., USA) equipped with a 30-m BPX70 columnessentially as described by Zhou et al. (2011) except that thetemperature ramping program was changed to initial temperature at 150°C., holding for 1 min, ramping 3° C./min to 210° C., then 50° C./min to240° C. for a final holding of 2 min. Peaks were quantified with AgilentTechnologies ChemStation software (Rev B.03.01 (317), Palo Alto, Calif.,USA). Peak responses were similar for the fatty acids of authenticNu-Check GLC standard-411 (Nu-Check Prep Inc, Minn., USA) whichcontained equal proportions of 31 different fatty acid methyl esters,including 18:1, 18:0, 20:0 and 22:0 was used for calibration. Theproportion of each fatty acid in the samples was calculated on the basisof individual and total peaks areas for the fatty acids.

Analysis of FAMES by Gas Chromatography—Mass Spectrometry

Confirming double bond positions in the FAME by 2,4-dimethyloxazoline(DMOX) modification and GC-MS analysis were carried out as previouslydescribed (Zhou et al., 2011), except with a Shimadzu GC-MS QP2010 Plusequipped with a 30-m BPX70 column. The column temperature was programmedfor an initial temperature at 150° C. for 1 min, ramping at 5° C./min to200° C. then 10° C./min to 240° C. with holding for 5 min. Mass spectrawere acquired and processed with GCMSsolution software (Shimadzu,Version 2.61). The free fatty acids and FAME standards were purchasedfrom Sigma-Aldrich (St. Louis, Mo., USA).

Analysis of Lipid Species by LC-MS

Mature individual single seeds were subjected to lipidomics analysisusing LC-MS at the School of Botany, University of Melbourne. Totallipids were extracted as described by Bligh and Dyer (1959) anddissolved in CHCl₃. Aliquots of one mg lipid were dried with N₂,dissolved in 1 mL of 10 mM butylated hydroxytoluene in butanol:methanol(1:1 v/v), and analysed using an Agilent 1200 series LC and 6410Belectrospray ionisation triple quadrupole LC-MS. Lipids werechromatographically separated using an Ascentis Express RP-Amide column(5 cm×2.1 mm, Supelco) and a binary gradient with a flow rate of 0.2mL/min. The mobile phases were: A, 10 mM ammonium formate inH₂O:methanol: tetrahydrofuran (50:20:30, v/v/v); B. 10 mM ammoniumformate in H₂O:methanol: tetrahydrofuran (5:20:75, v/v/v). Selectedneutral lipids (TAG and DAG) and phosphocholine (PC) with fatty acids16:0, 16:1 18:0, 18:1, 18:2, 18:3 were analysed by multiple reactionmonitoring (MRM) using a collision energy of 25 V and fragmentor of 135V. Individual MRM TAGs and DAGs were identified based on ammoniatedprecursor ion and product ion from neutral loss of fatty acid. TAGs andDAGs were quantified using the 10 uM tristearin external standard.

Analysis of the Sterol Content of Oil Samples

Samples of approximately 10 mg of oil together with an added aliquot ofC24:0 monol as an internal standard were saponified using 4 mL 5% KOH in80% MeOH and heating for 2 h at 80° C. in a Teflon-lined screw-cappedglass tube. After the reaction mixture was cooled, 2 mL of Milli-Q waterwere added and the sterols were extracted into 2 mL of hexane:dichloromethane (4:1 v/v) by shaking and vortexing. The mixture wascentrifuged and the sterol extract was removed and washed with 2 mL ofMilli-Q water. The sterol extract was then removed after shaking andcentrifugation. The extract was evaporated using a stream of nitrogengas and the sterols silylated using 200 mL of BSTFA and heating for 2 hat 80° C.

For GC/GC-MS analysis of the sterols, sterol-OTMSi derivatives weredried under a stream of nitrogen gas on a heat block at 40° C. and thenre-dissolved in chloroform or hexane immediately prior to GC/GC-MSanalysis. The sterol-OTMS derivatives were analysed by gaschromatography (GC) using an Agilent Technologies 6890A GC (Palo Alto,Calif., USA) fitted with an Supelco Equity™-1 fused silica capillarycolumn (15 m×0.1 mm i.d., 0.1 μm film thickness), an FID, asplit/splitless injector and an Agilent Technologies 7683B Series autosampler and injector. Helium was the carrier gas. Samples were injectedin splitless mode at an oven temperature of 120° C. After injection, theoven temperature was raised to 270° C. at 10° C. min⁻¹ and finally to300° C. at 5° C. min⁻¹. Peaks were quantified with Agilent TechnologiesChemStation software (Palo Alto, Calif., USA). GC results are subject toan error of ±5% of individual component areas.

GC-mass spectrometric (GC-MS) analyses were performed on a FinniganThermoquest GCQ GC-MS and a Finnigan Thermo Electron Corporation GC-MS;both systems were fitted with an on-column injector and ThermoquestXcalibur software (Austin, Tex., USA). Each GC was fitted with acapillary column of similar polarity to that described above. Individualcomponents were identified using mass spectral data and by comparingretention time data with those obtained for authentic and laboratorystandards. A full procedural blank analysis was performed concurrent tothe sample batch.

Quantification of TAG Via Iatroscan

One μl of each plant extract is loaded on one Chromarod-SII for TLC-FIDIatroscan™ (Mitsubishi Chemical Medience Corporation—Japan). TheChromarod rack is then transferred into an equilibrated developing tankcontaining 70 ml of a Hexane/CHCl₃/2-Propanol/Formic acid(85/10.716/0.567/0.0567 v/v/v/v) solvent system. After 30 min ofincubation, the Chromarod rack is then dried for 3 min at 100° C. andimmediately scanned on an Iatroscan MK-6_(S) TLC-FID analyser(Mitsubishi Chemical Medience Corporation—Japan). Peak areas of a DAGEinternal standard and the TAG are integrated using SIC-48011 integrationsoftware (Version:7.0-E SIC System instruments Co., LTD—Japan).

TAG quantification is carried out in two steps. First, the DAGE internalstandard is scanned in all samples to correct the extraction yieldsafter which concentrated TAG samples are selected and diluted. Next, theamount of TAG is quantified in diluted samples with a second scanaccording to the external calibration using glyceryl trilinoleate asexternal standard (Sigma-Aldrich).

Expression of Candidate FAD2 Genes in Saccharomyces cerevisiae

The DNA fragments containing the entire open reading frames of candidateFAD2 cDNAs were excised from pGEMT-easy vector as EcoRI fragments andinserted into the corresponding site of the vector pENTR11 (Invitrogen,Carlsbad, Calif., USA). The inserts were then cloned into thedestination expression vector pYES2-DEST52, to place the open readingframes under the control of the GAL1 promoter for inducible geneexpression in yeast cells, using the Gateway® Cloning recombinationtechnology (Stratagene, La Jolla, Calif., USA). The gene sequences inthe resultant plasmids were verified by DNA sequencing. The resultingplasmids and the pYES2-DEST52 vector lacking any cDNA insert as acontrol were introduced into cells of yeast Saccharomyces cerevisiaestrain YPH499 by lithium acetate-mediated transformation. Expression ofthese candidate FAD2 genes in yeast cells with or without exogenousfatty acid substrate feeding was essentially as previously described byZhou et al. (2006). Each experiment was carried out in triplicate.

Expression of Genes in Plant Cells in a Transient Expression System

Genes were expressed in Nicotiana benthamiana leaf cells using atransient expression system essentially as described by Voinnet et al.(2003) and Wood et al. (2009). A vector for constitutive expression ofthe viral silencing suppressor protein, P19, under the control of theCaMV 35S promoter was obtained from the laboratory of Peter Waterhouse,CSIRO Plant Industry, Canberra, Australia. The chimeric binary vector35S:P19 was introduced into Agrobacterium tumefaciens strain AGL1. Allother binary vectors containing a coding region to be expressed in theplant cells from a promoter, often the 35S promoter, were alsointroduced into A. tumefaciens strain AGL1. The recombinant cells weregrown to stationary phase at 28° C. in 5 mL LB broth supplemented with50 mg/L rifampicin and either 50 mg/L kanamycin or 80 mg/L spectinomycinaccording to the selectable marker gene on the binary vector. Thebacteria from each culture were pelleted by centrifugation at 3000×g for5 min at room temperature before being resuspended in 1.0 ml ofinfiltration buffer containing 5 mM MES, pH 5.7, 5 mM MgSO₄ and 100 μMacetosyringone. The resuspended cell cultures were then incubated at 28°C. with shaking for another 3 hours. A 10-fold dilution of each culturein infiltration buffer was then mixed with an equal volume of the35S:P19 culture, diluted in the same manner, and the mixturesinfiltrated into the underside of the fully expanded N. benthamianaleaves. Mixed cultures comprising genes to be expressed included the35S:P19 construct in Agrobacterium unless otherwise stated. Controlinfiltrations included only the 35S:P19 construct in Agrobacterium.

Leaves were infiltrated with the Agrobacterium cell mixtures and theplants were typically grown for a further five days after infiltrationbefore leaf discs were recovered for total lipid isolation and fattyacid analysis. N. benthamiana plants were grown in growth cabinets undera constant 24° C. with a 14/10 hr light/dark cycle with a lightintensity of approximately 200 lux using Osram ‘Soft White’ fluorescentlighting placed directly over plants. Typically, 6 week old plants wereused for experiments and true leaves that were nearly fully-expandedwere infiltrated. All non-infiltrated leaves were removed postinfiltration to avoid shading.

Real-Time Quantitative PCR (RT-qPCR)

Gene expression analysis was performed by quantitative RT-PCR using aBIORAD CFX96™ Real-time PCR detection system and iQ™ SYBR® GreenSupermix (BioRad, Hercules, Calif., USA). Primers of 19-23 nucleotidesin length and having a melting temperature (Tm) of about 65° C. and weredesigned for gene-specific amplifications that would result inamplification products of about 100-200 bp. PCR reactions were carriedout in 96-well plates. All RT-PCR reactions were performed intriplicate. The reaction mixture contained 1× iQ™ SYBR® Green Supermix(BioRad, Hercules, Calif., USA), 5 μM forward and reverse primers and400 ng of cDNA and was used at a volume of 10 uL per well. The thermalcycling conditions were 95° C. for 3 min, followed by 40 cycles of 95°C. for 10 s, 60° C. for 30 s and 68° C. for 30 s. The specificity of thePCR amplification was monitored by melting curve analysis following thefinal step of the PCR from 60° C. through 95° C. at 0.1° C./sec.Additionally, PCR products were also checked for purity by agarose gelelectrophoresis and confirmed by sequencing. The constitutivelyexpressed gene KASII was used as an endogenous reference to normaliseexpression levels. The data were calibrated relative to thecorresponding gene expression level following the 2^(−ΔΔCt) method forrelative quantification (Schmittgen, 2008). The data were presented asmeans±SD of three reactions performed on independent 96-well plates.

DNA Isolation and Southern Blot Analysis

Genomic DNA of safflower seedlings, genotype “SU”, was isolated fromfully expanded leaves using CTAB buffer and following the methoddescribed by Paterson et al. (1993). Further purification was carriedout using CsCl gradients as previously described (Liu et al., 1999).Aliquots of 10 μg of safflower genomic DNA were digested separately witheight different restriction enzymes, namely AccI, BglII, BamHI, EcoRI,EcoRV, HindIII, XbaI and XhoI. Genomic DNA digested with eachrestriction enzyme was electrophoresed through 1% agarose gels. The gelwas soaked in 0.5 M NaOH, 1.5 M NaCl for 30 min and the DNA blotted ontoa Hybond-N⁺ nylon membrane (Amersham, UK). The filters were probed withan α-P³² dCTP-labelled DNA fragment corresponding to the entire codingregion of the safflower CtFAD2-6 gene as a representative of the CtFAD2gene family at low stringency hybridization conditions. Thehybridizations were performed overnight at 65° C. in a solutioncontaining 6×SSPE, 10% Denhardt's solution, 0.5% SDS and 100 μg/mLdenatured salmon sperm DNA. Following the hybridisation and after abrief wash in 2×SSC/0.1% SDS at 50° C., the filters were washed threetimes, for 20 min each time, in 0.2×SSC/0.1% SDS at 50° C. prior toautoradiography.

Transformation of Safflower and Arabidopsis thaliana

Chimeric vectors comprising genes to be used to transform Arabidopsiswere introduced into A. tumefaciens strain AGL1 and cells from culturesof the transformed Agrobacterium used to treat A. thaliana (ecotypeColumbia) plants using the floral dip method for transformation (Cloughand Bent, 1998). Transformed safflower plants were produced as describedby Belide et al. (2011) using the transformed Agrobacterial cultures.

Example 2. Isolation of Safflower cDNAs which are Candidates forEncoding FAD2

Total RNA Extraction and cDNA Synthesis

In order to produce cDNA from safflower, total RNA was isolated from 100mg samples of frozen safflower tissues including developing embryos,leaves, roots and hypocotyls. This was done for each tissue separatelyusing an RNeasy® Plant total RNA kit (Qiagen, Hilden, Germany) accordingto the supplier's protocol. The RNA concentration in the preparationswas determined with a NanoDrop™ spectrophotometer ND1000 (Thermo FisherScientific, Victoria, Australia) and the RNA concentrations wereequalized before analysis. The quality and relative quantities of theRNA in each preparation were visualized by gel electrophoresis ofsamples through 1% (w/v) agarose gels containing formaldehyde. The RNApreparations were treated with RQ1 RNase-free DNase (Qiagen, Hilden,Germany) to remove contaminating genomic DNA. First-strand cDNA wassynthesized from 400 ng of each DNA-free RNA preparation using theSuperScript III First-Strand Synthesis System (Qiagen, Hilden, Germany)with oligo(dT)₂₀ primer, following the manufacturer's instructions.

Isolation of Seed-Expressed FAD2 cDNAs from a Developing Seed cDNALibrary

Initially, the seed expressed safflower FAD2 cDNAs were obtained byscreening a cDNA library derived from developing embryos of safflowergenotype “SU” (wild-type, high linoleic acid levels). Libraryconstruction began with RNA extraction from a mixture of immatureembryos of different developmental stages which were harvested andground to powder in liquid nitrogen and RNA extraction was carried outusing TRIzol following the manufacturer's instruction (Invitrogen,Carlsbad, Calif., USA). Poly(A)-containing RNA was isolated using aQiagen mRNA purification kit (Qiagen, Hilden, Germany).

First strand oligo(dT)-primed cDNA was synthesised and converted todouble stranded DNA using a Stratagene cDNA synthesis kit, according tothe manufacturer's instructions (Stratagen, La Jolla, Calif., USA). Theblunt-ended cDNA was ligated with EcoRI adaptors, phosphorylated, andsize fractionated by gel-filtration in a Chroma spin+TE-400 column(Clontech, Calif., USA). The recombinant cDNAs were propagated in the E.coli strain XL-1 Blue MRF′ using a Stratagene Predigested Lambda ZAPII/EcoRI/CIAP cloning kit.

To identify the FAD2 clones, the library was screened using a DNAfragment corresponding to the coding region of Arabidopsis FAD2 (GenBankaccession no. L26296), following the protocol previously described (Liuet al., 1999). Positive plaques were purified through two successiverounds of screening and the purified phagemids containing putative FAD2cDNAs were excised as outlined in the Stratagene λZAPII cDNA SynthesisKit instruction manual. Sequence analysis of the FAD2 sequences weredone by the NCBI Blast program (www.ncbi.nlm.nih.gov/BLAST/). The openreading frame was predicted by using VectorNTI. Two different fulllength cDNAs were isolated from developing seed cDNA library and namedas CtFAD2-1 and CtFAD2-2, respectively.

Identification of ESTs for Candidate FAD2 Genes

To identify additional candidate FAD2 cDNAs, the Compositae GenomeProject (CGP) expressed sequence tag (EST) database of safflower(cgpdb.ucdavis.edu/cgpdb2.) was interrogated using the program BLASTpfor ESTs that encoded polypeptides having similarity with the A.thaliana FAD2 (GenBank accession No. L26296). At least eleven distinctFAD2 cDNA sequence contigs were identified, among which two contigsshowed identical sequences with CtFAD2-1 and CtFAD2-2 isolated fromsafflower seed cDNA library. In addition, nine different cDNAs wereidentified and designated as CtFAD2-3 through to CtFAD2-11,respectively.

3′- and 5′ RACE

The longest EST clone of each of the 9 contigs (CtFAD2-3 through toCtFAD2-11) was selected as the starting point for isolation of thecorresponding full length cDNA sequences. The process used 3′- and5′-Rapid Amplification of cDNA Ends (RACE) using cDNA produced from RNAsobtained from various safflower tissues including developing embryos,leaves, roots, hypocotyls and flowers. Gene specific primers (GSP) weredesigned from the longest EST clone of each contig. 3′-RACE wasperformed using a one-step RT-PCR kit following the manufacturer'sinstructions (Bioline, London, UK). A gene-specific primer (GSP) wasused in a first round of PCR amplification for each of the selected ESTsin combination with a poly(dT) primer with a NotI site at its 3′ end. Asecond round of PCR was performed on the product of the first roundusing a nested GSP in combination with the poly(dT) primer. GSPs for 3′RACE are listed in Table 2.

Cloning of the 5′ end of the CtFAD2-6 cDNA was performed with 5′ RACESystem Kit (Invitrogen, Carlsbad, Calif., USA). Only the CtFAD2-6 mRNAwas reverse transcribed to cDNA using a gene-specific primer GSP1,5′-ACCTAACGACAGTCATGAACAAG-3′ (SEQ ID NO: 76). A nested gene-specificprimer GSP2, 5′-GTGAGGAAAGCGGAGTGGACAAC-3′ (SEQ ID NO: 77) was used inthe first PCR amplification. The reaction conditions used a hot start at95° C. for 4 min before adding the polymerase, 33 cycles of denaturationat 94° C. for 45 s, annealing at 55° C. for 1 min and extension at 72°C. for 2 min.

The amplified 3′ and 5′ fragments were subcloned into the vectorpGEM-Teasy and sequenced from both directions. Sequence comparisons ofthe 3′ and 5′ ends of the cloned fragments with the corresponding ESTsshowed overlapping regions that matched with each other, therebyproviding the 3′ and 5′ sequences for each gene and allowing theassembly of a putative full length sequence for each of the 11 cDNAs.

Isolation of Full Length cDNA Sequences for the Candidate CtFAD2 Genes

To isolate full length protein coding regions for the nine CtFAD2 genes,the ORFs were amplified using the One-step RT-PCR kit using total RNAsderived from several safflower tissues including developing embryos,leaves, roots, hypocotyls and flowers (Stratagene, La Jolla, Calif.,USA). The primers (Table 3) used to amplify the ORFs were based on theDNA sequences located in the 5′ and 3′ UTR of each cDNA. The amplifiedPCR products were cloned to vector pGEM-Teasy®, and their nucleotidesequences obtained by DNA sequencing.

Characteristics of the Candidate FAD2 Sequences from Safflower

Characteristics of the 11 cDNAs are summarised in Table 4 and of thepolypeptides in Table 5.

The predicted amino acid sequences of the encoded polypeptides CtFAD2-1to CtFAD2-11 shared extensive sequence identity, from about 44% to 86%identity with each other. They showed 53% to 62% sequence identity withArabidopsis FAD2. The sizes of the predicted polypeptides were in therange from 372 to 388 amino acids, that is, they were all about 380aaresidues in length. The cDNAs had unique 5′ and 3′ untranslated region(UTR) sequences, therefore the endogenous genes could readily berecognised by their UTR sequences. Amplification of the protein codingregions from safflower genomic DNA resulted in identical DNA sequenceswith the corresponding cDNA for each of the 11 genes, indicating thatthere were no introns interrupting their protein-coding regions.

TABLE 2 Oligonucleotide primers used in the 3′RACE of multiple FAD2genes in safflower. Primer gene Sense sequence Antisense sequenceCtFAD2-3 5′- 5′- CTTCAGCGAGTACCAATGG GGTTTCATCGTCCACTCCTTCTCGAC-3′ (SEQ ID NO: 58) GA -3′ (SEQ ID NO: 59) CtFAD2-4 5′- 5′-CTTCAGCGAGTACCAATGG GGTTTCATCGTCCACTCCTT CTCGAC-3′ (SEQ ID NO: 60)GA -3′ (SEQ ID NO: 61) CtFAD2-5 5′- 5′- ATGACACCATTGGCTTCATCTTTCTGCTCACTCCATACT CTGCCA -3′ (SEQ ID NO: 62) TC -3′ (SEQ ID NO: 63)CtFAD2-6 5′- 5′- AGCGAATATCAGTGGCTTG ACTCCGCTTTCCTCACTCCGACGATG -3′ (SEQ ID NO: 64) TAC -3′ (SEQ ID NO: 65) CtFAD2-7 5′- 5′-CATGAATGTGGTCATCATG CTTCTTCATCCATTCGGTTT CCTTTAG -3′ (SEQ ID NO: GC -3′ (SEQ ID NO: 67) 66) CtFAD2-8 5′- 5′- CGTGGTTGAATGACACCATACCTTCTACACACCGGTAT TGGTTAC -3′ (SEQ ID NO:  GCCT -3′ (SEQ ID NO:  68)69) CtFAD2-9 5′- 5′- CATGGAAGATAAGCCACCG AACACGGGTTCGCTTGAGCTCGACATC -3′ (SEQ ID NO: ACGA -3′ (SEQ ID NO: 70) 71) CtFAD2-10 5′- 5′-TGCATACCCGCAAGCAAAA CCATCTCTCGAGAGTTCCT CCG -3′ (SEQ ID NO: 72)TAC -3′ (SEQ ID NO: 73) CtFAD2-11 5′- 5′- ATGTGGTCACCATGCCTTTTGGAATGGTCCTCCATTCC AGTGAG -3′ (SEQ ID NO:  GCTC -3′ (SEQ ID NO:  74)75)

TABLE 3 Oligonucleotide primers used for amplification of the entirecoding region of FAD2 genes in safflower. Primer gene Sense sequenceAntisense sequence CtFAD2-1 5′- 5′- TGAAAGCAAGATGGGAGGTCACAACTTTACTTATTCTTG AGG -3′ (SEQ ID NO: 78) T -3′ (SEQ ID NO: 79)CtFAD2-2 5′- 5′- ATTGAACAATGGGTGCAG CATCATCTTCAAATCTTATTCGC -3′ (SEQ ID NO: 80) -3′ (SEQ ID NO: 81) CtFAD2-3 5′- 5′-AATCAGCAGCAGCACAAG CAAACATACCACCAAATGCT C -3′ (SEQ ID NO: 82)ACT -3′ (SEQ ID NO: 83) CtFAD2-4 5′- 5′- CTCAGTAACCAGCCTCAAAGCGGATTGATCAAATACTTG ACTTG -3′ (SEQ ID NO: 84) TG -3′ (SEQ ID NO: 85)CtFAD2-5 5′- 5′- ATCACAGGAAGCTCAAAG GTAGGTTATGTAACAATCGTCATCT -3′ (SEQ ID NO: 86) G -3′ (SEQ ID NO: 87) CtFAD2-6 5′- 5′-TGAAGACGTTAAGATGGG GTAGGTTATGTAACAATCGT AGCTG -3′ (SEQ ID NO: 88)G -3′ (SEQ ID NO: 89) CtFAD2-7 5′- 5′- CAGATCCAACACTTCACCAAGATCTAAAGAATTTCCATG CCAG -3′ (SEQ ID NO: 90) GTG -3′ (SEQ ID NO: 91)CtFAD2-8 5′- 5′- CTGCTCTCTACGACACTAA TCTATCTAATGAGTATCAAGATTCAC -3′ (SEQ ID NO: 92) GAAC -3′ (SEQ ID NO: 93) CtFAD2-9 5′- 5′-CTGAATTCACACCCACAGA ACATCCCTTCTTAGCTTTAA TAGCTAG -3′ (SEQ ID NO:CTA-3′ (SEQ ID NO: 95) 94) CtFAD2-10 5′- 5′- ACTTCGCCCTCTGTTATCTCCATACACATACATCCTACA GG -3′ (SEQ ID NO: 96) CGAT -3′ (SEQ ID NO: 97)CtFAD2-11 5′- 5′- ACTCACAATAACTTCATCT CTACTAGCCATACAATGTCTCTCTC -3′ (SEQ ID NO: 98) TCG -3′ (SEQ ID NO: 99)

TABLE 4 Characteristics of the candidate FAD2 cDNAs from safflower. GenecDNA Protein coding Size of Size of Position Size of ORF nucleotidedesignation length (nt) region 5′UTR 3′UTR of intron intron SEQ ID NO.CtFAD2-1 1422 124-1266  123 156 −13 1144 12 CtFAD2-2 1486 81-1233 80 253−12 3090 13 CtFAD2-3 1333 51-1197 50 136 −11 114 14 CtFAD2-4 140352-1227 51 176 −11 124 15 CtFAD2-5 1380 66-1194 65 186 −33 122 16CtFAD2-6 1263 15-1146 14 117 * * 17 CtFAD2-7 1375 66-1185 65 190 −29 25318 CtFAD2-8 1345 58-1207 57 138 * * 19 CtFAD2-9 1326 108-1172  107154 * * 20 CtFAD2-10 1358 56-1199 55 159 −38 2247 21 CtFAD2-11 122958-1092 57 137 −22 104 22

TABLE 5 Characteristics of candidate CtFAD2 polypeptides. PolypetidePosition (& Amino length (No. Position (& sequence) of Position (& acidGene of amino sequence) of second His sequence) of SEQ ID designationacids) first His box box third His box NO. CtFAD2-1 380 105 HECGH*141 HRRHH 315 HVVHH 27 CtFAD2-2 384 106 HECGH 142 HRRHH 316 HVTHH 28CtFAD2-3 381 104 HECGH 140 HRTHH 314 HAVHH 29 CtFAD2-4 380 103 HECGH139 HRTHH 313 HAVHH 30 CtFAD2-5 375 102 HDCGH 138 HRTHH 311 HVVHH 31CtFAD2-6 376 101 HDLGH 137 HRSHH 310 HVVHH 32 CtFAD2-7 372  99 HECGH135 HRTHH 308 HAVHH 33 CtFAD2-8 382 103 HECGH 139 HRTHH 313 HAVHH 34CtFAD2-9 387 107 HECGH 143 HRTHH 318 HAVHH 35 CtFAD2- 380 104 HECGH140 HRRHH 314 HVVHH 36 10 CtFAD2- 377 100 HECGH 136 HRNHH 310 HVLHH 3711 *HECGH (SEQ ID NO: 159), HDCGH (SEQ ID NO: 160), HDLGH (SEQ ID NO:161), HRRHH (SEQ ID NO: 162), HRTHH (SEQ ID NO: 163), HRSHH (SEQ ID NO:164), HRNHH (SEQ ID NO: 165), HVVHH (SEQ ID NO: 166), HVTHH (SEQ ID NO:167), HAVHH (SEQ ID NO: 168), and HVLHH (SEQ ID NO: 169).

To investigate the relationship of the safflower candidate FAD2polypeptides to known FAD2 enzymes, the 11 deduced polypeptide sequenceswere aligned with plant FAD2 sequences and a neighbour-joining tree wasconstructed using Vector NTI (FIG. 1). As shown in FIG. 1, the aminoacid sequences of CtFAD2-1 and CtFAD2-10 were most closely related,first of all to each other and then to seed expressed FAD2s from otherspecies. CtFAD2-2 was more closely related to constitutively expressedgenes from other species than to other candidate FAD2s in safflower.CtFAD2-3, -4, -5, -6 and -7 formed a new branch in the neighbour-joiningtree, most likely as the evolutionary result of a recently diverged genebecoming multiplied in safflower. Interestingly, in relatedness to otherspecies, these were most closely related to a functionally divergentFAD2 conjugase from Calendula officinalis. FAD2-11 was more closelyrelated to acetylenases from several plant species, including thesunflower vFAD2 which was induced by fungal elicitors (Cahoon et al.,2003). It appeared that CtFAD2-8 and -9 were more divergent than theother candidate FAD2s from safflower. However, this analysis also showedthat the sequence comparisons, although they gave some hints aboutpossible function, could not by themselves provide reliable conclusionsabout the function of the different FAD2 candidates. Therefore,functional analysis was required to make conclusions about the functionof each gene/polypeptide.

The sequence comparisons showed that the safflower candidate FAD2polypeptides shared about 50%-60% sequence identity and 52%-65%similarity to known FAD2 enzymes from other species. The extent of DNAsequence divergence among the safflower CtFAD2 genes reflected theirphylogenetic relationships, in that CtFAD2-3, -4 and -5 are all moresimilar to each other than to CtFAD2-1, or CtFAD2-10, and vice versa.These numbers have close parallels in the amino acid identity matrix(Table 6).

TABLE 6 Sequence identity of the coding region DNA and deduce aminoacids in safflower FAD2 genes. Deduced amino acid identity (%) CtFA CtFACtFA CtFA CtFA CtFA CtFA CtFA CtFA CtFA CtFA D2-1 D2-2 D2-3 D2-4 D2-5D2-6 D2-7 D2-8 D2-9 D2-10 D2-11 CtFA — 70.3 53.2 52.5 53.5 50.9 54.159.7 59.5 80.1 56.4 D2-1 CtFA 70.0 — 54.5 55.0 54.2 51.7 57.8 60.6 62.569.5 58.6 D2-2 CtFA 62.0 62.0 — 97.1 62.0 61.8 63.1 52.7 50.9 51.2 56.8D2-3 CtFA 62.7 63.3 95.1 — 61.4 61.4 63.3 53.1 50.9 50.9 56.9 D2-4 CtFA61.9 60.3 69.7 70.6 — 63.2 62.0 51.4 51.3 51.7 53.9 D2-5 CtFA 60.6 59.968.8 69.6 72.0 — 63.1 49.3 50.8 50.1 56.2 D2-6 CtFA 62.2 65.8 69.4 69.366.6 68.2 — 51.7 49.2 51.4 60.7 D2-7 CtFA 65.2 66.2 63.1 62.8 60.8 61.761.2 — 58.8 58.1 56.40 D2-8 CtFA 64.9 66.2 59.5 59.5 58.3 59.2 59.5 63.5— 59.3 55.9 D2-9 CtFA 78.9 72.0 60.7 62.0 59.8 61.0 60.7 64.3 64.1 —57.2 D2-10 CtFA 60.0 62.9 64.1 64.4 62.4 64.1 62.7 63.9 60.9 61.7 —D2-11Characteristics of the Candidate CtFAD2 Polypeptides

The predicted polypeptides of the 11 candidate CtFAD2s each contained anaromatic amino acid-rich motif at the very end of the C-terminus. Suchmotifs have been identified in other plant FAD2 polypeptides, and arethought to be necessary for maintaining localization in the ER(McCartney et al., 2004). Consistent with other plant membrane boundfatty acid desaturase enzymes, the predicted CtFAD2 polypeptides eachcontained three histidine-rich motifs (His boxes). Such His-rich motifsare highly conserved in FAD2 enzymes and have been implicated in theformation of the diiron-oxygen complex used in biochemical catalysis(Shanklin et al., 1998). In most of the candidate CtFAD2 polypeptidesequences, the first histidine motif was HECGHH, the exceptions beingCtFAD2-5 and -6 which had HDCGHH and HDLGHH, respectively. The lastamino acid of the first His box in CtFAD2-8 (HECGHQ) was a Q rather thana H. The inventors looked for this motif in 55 known plant FAD2 enzymesand the H to Q substitution is also present in a diverged FAD2 homologuefrom Lesquerella lindheimeri with predominantly fatty acid hydroxylaseactivity (Genbank Accession number EF432246; Dauk et al., 2007). Thesecond histidine motif was highly conserved, as the amino acid sequenceHRRHH, in several candidate safflower FAD2s, including CtFAD2-1, -2, -8,-9 and -10. It was noteworthy that the amino acid N was found inCtFAD2-11 at the +3 position of the motif, which was also seen in anumber of functionally divergent FAD2-type enzymes including Crepisalpina CREP1, Crepis palaestina Cpal2 and sunflower vFAD2 (AY166773.1),Calendula officinalis FAC2 (AF343064.1), Rudbeckia hirta acetylenase(AY166776.1). The amino acid at this position in CtFAD2-3, -4, -5, -6and -7 was either an S or a T.

In each of the CtFAD2-1, -2, -9 and -10 polypeptides, the amino acidimmediately preceding the first histidine box was an alanine, the sameas for other plant fatty acid Δ12-desaturase enzymes. The amino acidvaline (V) rather than alanine was present at that position in CtFAD2-5,while the other six CtFAD2 polypeptides had a glycine in this position.It was proposed by Cahoon et al. (2003) that a glycine substitution foralanine at this position has been found in functionally divergent FAD2enzymes, except fatty acid Δ12-hydroxylase. As described in thefollowing Examples, subsequent heterologous expression experimentstesting the function of the candidates demonstrated that each of theCtFAD2-1, -2 and -10 polypeptides were oleate Δ12-desaturases, whileCtFAD2-9 showed desaturase specificity to palmitoleate (C16:1) ratherthan oleate.

It was noted that of the 11 candidate CtFAD2s, only the CtFAD2-11polypeptide had a DVTH sequence in the −5 to −2 positions of the thirdHistidine box, which was consistent with the (D/N)VX(H/N) motif proposedto occur in all plant acetylenases (Blacklock et al., 2010). The fiveamino acids immediately after the third histidine box of the CtFAD2-1,-2 and -10 polypeptides were LFS™, as for other known plant FAD2 oleatedesaturases. In contrast, CtFAD2-9, the palmitoleate specific fatty aciddesaturase, had a LFSYI motif at this position with two amino acidsubstitutions at +4 and +5 position. In the CtFAD2-3, -4 and -5polypeptides, the S at the +3 position was substituted by P, which wasalso present exclusively in other FAD2 fatty acid conjugases includingthose from Calendula officinalis (FAC2, accession AAK26632) andTrichosanthes kirilowii (accession AAO37751).

It has been shown that the Serine-185 of the soybean FAD2-1 enzyme isphosphorylated during seed development as a regulatory mechanism for itsenzymatic activity (Tang et al., 2005). Among the 11 candidate CtFAD2polypeptides, only CtFAD2-1 had a serine in the corresponding position(Serine-181) relative to soybean FAD2-1. It was concluded that the sameposttranslational regulatory mechanism might operate during safflowerseed development and oil accumulation through phosphorylation of theserine-185, to modulate microsomal Δ12 oleate desaturation in thedeveloping seed.

Example 3. Isolation of Genomic Sequences for FAD2 Candidates

Isolation of 5′UTR Introns of the Candidate CtFAD2 Genes

The intron-exon structures of FAD2 genes are conserved in many floweringplants. All FAD2 genes studied so far contain only one intron which islocated at the 5′UTR, with one exception being soybean FAD2-1 for whichthe intron is located in the coding region immediately following thefirst ATG, the translational initiation codon (Liu et al., 2001; Kim etal., 2006; Mroczka et al., 2010). Intron sequence divergence could beused as a measure of evolutionary distance between taxonomically closelyrelated species (Liu et al., 2001).

In order to isolate the DNA sequences of possible introns situatedwithin the 5′-UTRs of the candidate CtFAD2 genes, the typical intronsplice sites (AG:GT) were predicted in the 5′ UTR of each CtFAD2 cDNAsequence, and PCR primers were designed based on the flanking sequencesof predicted splice sites. The primers are listed in Table 7. GenomicDNA isolated from safflower genotype SU was used as template in PCRreactions to amplify the genomic regions corresponding to the 5′UTRs.The amplifications were accomplished in 50 μL reactions with 100 ng ofgenomic DNA, 20 pmol of each primer and a Hotstar (Qiagen, Hilden,Germany) supplied by the manufacturer. PCR temperature cycling wasperformed as follows: 94° C. for 15 min for one cycle, 94° C. for 30 s,55° C. for 1 min, 72° C. for 2 min for 35 cycles; 72° C. for 10 minusing the Kyratec supercycler SC200 (Kyratec, Queensland, Australia).The PCR products were cloned into pGEM-T Easy and then sequenced.

The inventors were able to obtain the predicted 5′ intron from 8 of the11 candidate CtFAD2 genes, namely CtFAD2-1, -2, -3, -4, -5, -7, -10 and-11. The major features of these introns are given in Table 8. Theintron was not amplified successfully from CtFAD2-6, -8 and -9, probablydue to an insufficient length of the 5′ UTR in which the introns werepresent. It appeared that an intron-less FAD2 has not been reported,although intron loss from nuclear genes has been commonly observed inhigher plants (Loguercio et al., 1998; Small et al., 2000a;b).

TABLE 7 Oligonucleotide primers used for the amplification of 5′UTRregions ofc andidate FAD2 genes in safflower. Primer gene Sense sequenceAntisense sequence CtFAD2-1 5′- 5′- GAGATTTTCAGAGAGCAACTTTGGTCTCGGAGGCAGAC GCGCTT -3′(SEQ ID NO: ATA -3′(SEQ ID NO: 101) 100)CtFAD2-2 5′- 5′- CAAAAGGAGTTTCAGAAA ACTCGTTGGATGCCTTCGAGGCCTCC -3′(SEQ ID NO: TTC- 3′(SEQ ID NO: 103) 102) CtFAD2-3 5′ 5′-AATCAGCAGCAGCACAAG AAGGCGGTGACAATTATGA C -3′(SEQ ID NO: 104)TATC -3′(SEQ ID NO: 105) CtFAD2-4 5′- 5′- CTCAGTAACCAGCCTCAAAAGGCGGAGACGATTATGA AACTTG -3′(SEQ ID NO: TATC -3′(SEQ ID NO: 107) 106)CtFAD2-5 5′- 5′- ATCACAGGAAGCTCAAAG ATCATCTCTTCGGTAGGTTACATCT -3′(SEQ ID NO: 108) TG -3′(SEQ ID NO: 109) CtFAD2-7 5′- 5′-CAGATCCAACACTTCACCA CTAAAGAATTTCCATGGTGT CCAG -3′(SEQ ID NO: 110)TAC -3′(SEQ ID NO: 111) CtFAD2-10 5′- 5′- ACTTCGCCCTCTGTTATCTGAGAGACGGTGGAAGTAGG GG -3′(SEQ ID NO: 112) TG -3′(SEQ ID NO: 113)CtFAD2-11 5′- 5′- CTCACAATAACTTCATCTC AAAGACATAGGCAACAACGTCTC -3′(SEQ ID NO: 114) AGATC -3′(SEQ ID NO: 115)

TABLE 8 The feature of candidate FAD2 gene introns. Feature CtFAD2-1CtFAD2-2 CtFAD2-3 CtFAD2-4 CtFAD2-5 CtFAD2-7 CtFAD2-10 CtFAD2-11Position  -13  -12 -11 -11 -33 -29  -38 -22 Length 1144 3090 114 124 122253 2247 104 AT 64.5% 65.8% 73.7% 75.0% 67.2% 62.1% 68.9% 75% content CG35.5% 34.2% 26.3% 25.0% 32.8% 37.9% 31.1% 25% content 5′E/I AG:GTGCATAG:GTGAGA AG:GTATGA AG:GTAAGT AG:GTGAAG AG:GTATAC TG:GTTCGT AG:GTTTCTboundary 3′I/E TTGCAG:GT TTGCAG:GT ATGCAG:GT GCGCAG:GT TTTCAG:GTTTGCAG:GT ATATAG:GT TTGCAG:GT boundary

The intron sequence in each of the eight genes was located within the5′-UTR of each gene, at positions that ranged from 11 to 38 bp upstreamof the putative translation start codon, the first ATG in each openreading frame. The intron length ranged from 104 bp (CtFAD2-11) to 3,090bp (CtFAD2-2) (Table 8). For CtFAD2-1, the intron size was 1,144 bp,similar in size to introns identified in FAD2 genes from Arabidopsis(The Arabidopsis Information Resource, www.arabidopsis.org), cotton (Liuet al., 2001) and sesame (Sesamum indicum) (Kim et al., 2006). Thedinucleotides at the putative splice sites, AG and GT, were conserved inall eight of the examined CtFAD2 genes, but otherwise the intronsequences were all divergent in sequence without any significanthomology between them. The intron sequences were all A/T-rich with anA/T content of between 61% and 75%, which was consistent with many otherintron sequences from dicotyledonous plants. In genes from other dicotplants, the Arabidopsis FAD2 gene had a 1,134-bp intron just 5 bpupstream from its ATG translation initiation codon. The size of the5′-UTR intron of the Gossypium FAD2-1 gene was 1,133 bp, located 9 bpupstream from the translation initiation codon. In contrast, the cottonFAD2-4 and FAD2-3 genes had larger 5′-UTR introns of 2,780 bp and 2,967bp, respectively, located 12 bp upstream from the translation startcodon. Each candidate CtFAD2 gene could be distinguished by thedifferences in the position and size of the 5′-UTR intron in each gene.The differences could also be important in providing for differentialexpression of the genes. Such introns have been reported to havepositive effects on the expression of reporter genes in sesame (Kim etal., 2006). A corresponding intron was shown to be an effective targetfor posttranscriptional gene silencing of FAD2 in soybean (Mroczka etal., 2010).

It has been known that introns may have dramatic effects on geneexpression profiles. Analyzing the intron sequences by the PLACE program(www.dna.affrc.go.jp/PLACE/) identified several putative cis-regulatoryelements. For instance, a few motifs, such as ABRE and SEF4, commonlypresent in the seed-specific promoters have been located in theseed-specific CtFAD2-1. An AG-motif which is normally found in thepromoter of defence-related genes induced by various stresses such aswounding or elicitor treatment was located at CtFAD2-3 that isspecifically expressed in the hypocotyls and cotyledons of saffloweryoung seedlings.

Example 4. Southern Blot Hybridisation Analysis of the CandidateSafflower FAD2 Genes

The complexity of the FAD2-like gene family in safflower was examined bySouthern Blot hybridisation analysis. Low stringency hybridisationanalysis showed that, in safflower, FAD2 was encoded by a complexmultigene family (FIG. 2). By counting the hybridising fragmentsobtained by using various restriction enzymes to cleave the genomic DNA,it was estimated that there were more than 10 FAD2 or FAD2-like genes insafflower. The differences seen in the intensity of hybridization forthe different fragments presumably correlated with the relative levelsof sequence identity to the probe DNA that was used. Safflower is adiploid species and is thought to have a single wild progenitor species,C. palaestinus (Chapman and Burke, 2007). The inventors speculate thatthe unusually large FAD2 gene family in safflower is perhaps derivedfrom some ancient gene duplications, leading to specialisation anddifferential activity of the different members of the gene family.

Example 5. Functional Analysis of Candidate Genes in Yeast and PlantCells

Expression of Candidate CtFAD2 Genes in Yeast—Functional Analysis

As a convenient host cell, the yeast S. cerevisiae has been used forstudying the functional expression of several plant FAD2 Δ12 oleatefatty acid desaturases (Covello and Reed 1996; Dyer et al., 2002;Hernández et al., 2005). S. cerevisiae has a relatively simple fattyacid profile and it contains ample oleic acid in its phospholipid whichcan be used as a substrate for FAD2 enzymes. It also lacks an endogenousFAD2 activity. Therefore, the 11 candidate CtFAD2 genes were tested inyeast strain YPH499 using pYES2 derived constructs, each open readingframe under the control of the GAL1 promoter, as described in Example 1.

As shown in FIG. 3, when the fatty acid composition of yeast cellscontaining the “empty vector” pYES2 was analysed, no linoleic acid(18:2) or hexadecadienoic acid (16:2) was detected, as expected sinceyeast lacks endogenous FAD2. In contrast, the gas chromatogram for fattyacids obtained from yeast cells expressing the CtFAD2-1, CtFAD2-2 andCtFAD2-10 open reading frames each showed a fatty acid peak with aretention time of 11.293 min, corresponding to linoleic acid (C18:2),and the gas chromatograms for CtFAD2-9 and CtFAD2-10 showed a fatty acidpeak with retention time of 8.513 min, corresponding to C16:2. Thesedata indicated that CtFAD2-1, CtFAD2-2 and CtFAD2-10 were able toconvert oleic acid to linoleic acid and therefore were Δ12 oleatedesaturases. However, the level of 18:2 produced was lower than for theArabidopsis AtFAD2 construct which was used as positive control.CtFAD2-10 produced both linoleic acid (C18:2) and hexadecadienoic acid(C16:2) using oleic acid (C18:1) and palmitoleic acid (C16:1) assubstrates, respectively, while CtFAD2-9 desaturated palmitoleic acidand was therefore a Δ12 palmitoleate desaturase. Two minor new peaksthat appeared in the chromatograms of FAMEs from yeast cells expressingCtFAD2-11 were identified as linoleic acid (18:2^(Δ9(Z),12(Z))) and itstrans isomer (18:2^(Δ9(Z),12(E))) by GC-MS of their pyrrolidide adducts,and DMOX (FIG. 3H). Table 9 summaries the fatty acid composition ofyeast cells expressing CtFAD2 coding regions. No new peaks were detectedin yeast cells expressing CtFAD2-3, -4, -5, -6, -7 and -8.

To examine whether any of the candidate CtFAD2 polypeptides had fattyacid hydroxylase activity, FAMEs prepared from the yeast cellsexpressing each of the CtFAD2 open reading frames were reacted with asilylating reagent that converts hydroxyl residues into TMS-etherderivatives from which the mass spectra could be examined. However, nohydroxyl derivatives of the common fatty acids such as oleic acid weredetected in any of the yeast cell lines expressing the candidate CtFAD2open reading frames. This indicated that none of the 11 CtFAD2 genesencoded polypeptides having fatty acid hydroxylase activity in yeast.

Additional experiments were carried out to detect Δ12-epoxygenase andΔ12-acetylenase activity, both of which use linoleic acid as the fattyacid substrate, by supplementing the growth media of the same yeast celllines with free linoleic acid and analysing the fatty acid compositionafterward. The supplementation was done after addition of galactose tothe cultures to express the constructs. No novel fatty acid peaks weredetected in the gas chromatograms, including those representing epoxyand acetylenic fatty acid derivatives. The heterologous expression ofthese novel fatty acids in yeast, with supplementation of exogenous freefatty acids, has encountered some difficulties in demonstrating activity(Lee et al., 1998; Cahoon et al., 2003). Therefore, functional analysesin plant cells were carried out as follows.

TABLE 9 Fatty acid composition of yeast cells expressing selected CtFAD2genes. C14:0 C14:1 C16:0 C16:1 C16:2 Vector 1.30 ± 0.15 0.31 ± 0.0623.62 ± 1.62 36.04 ± 1.77 CtFAD2-1 1.17 ± 0.02 0.31 ± 0.01 22.96 ± 0.0437.15 ± 0.16 0.28 ± 0.02 CtFAD2-2 1.17 ± 0.06 0.29 ± 0.01 22.02 ± 0.4636.60 ± 0.07 CtFAD2-9 1.13 ± 0.06 0.18 ± 0.01 21.30 ± 0.59 34.32 ± 0.541.61 ± 0.09 CtFAD2-10 1.04 ± 0.01 0.27 ± 0.02 22.31 ± 0.03 34.79 ± 0.211.23 ± 0.03 CtFAD2-11 0.63 ± 0.01 0.17 ± 0.00 18.41 ± 0.34 37.27 ± 0.16C18:0 C18:1 C18:1^(Δ11) C18:2 C18:2^(Δ9Z, 12E) Vector 7.69 ± 0.74 29.62± 0.99 1.42 ± 0.20 CtFAD2-1 7.37 ± 0.12 26.36 ± 0.24 1.57 ± 0.02 2 82 ±0.14 CtFAD2-2 7.38 ± 0.07 30.95 ± 0.51 1.48 ± 0.04 0.11 ± 0.01 CtFAD2-98.90 ± 0.13 31.29 ± 0.25 1.27 ± 0.08 CtFAD2-10 8.08 ± 0.09 25.43 ± 0.071.34 ± 0.01 5.49 ± 0.09 CtFAD2-11 7.52 ± 0.07 33.25 ± 0.26 1.91 ± 0.010.32 ± 0.02 0.51 ± 0.03 (n = 3)Transient Expression of Candidate CtFAD2 Genes in N. benthamiana

To express the genes in a constitutive fashion in plant cells, inparticular in plant leaves, each of the CtFAD2 ORFs was inserted in thesense orientation into a modified pORE04 binary vector between theenhanced CaMV-35S promoter and the nos3′ terminator containing apolyadenylation signal sequence (Coutu et al., 2007) (SEQ ID NO: 54).Previous research indicated that the expression of transgenes could besignificantly enhanced by the co-expression of the viral silencingsuppressor protein, P19, to reduce host transgene silencing in a N.benthamiana leaf-based transient assay (Voinnet et al., 2003; Wood etal., 2009; Petrie et al., 2010). These experiments were performed asdescribed in Example 1.

As described above, the function of CtFAD2-11 was initially assessed byexpression in S. cerevisiae and two novel fatty acids were identified byGC-MS as 18:2^(Δ9(Z),12(Z)) and 18:2^(Δ9(Z),12(E)), respectively.Consistent with the results obtained from yeast, expression of CtFAD2-11in N. benthamiana leaves yielded a novel 18:2 trans isomer. The methylester of this isomer displayed a GC retention time that was identical tothat of a methyl 18:2^(Δ9(Z),12(E)) (FIG. 4B). The novel18:2^(Δ9(Z),12(E)) accounted for 0.35% of the fatty acids in leavesafter transiently expressing CtFAD2-11 (Table 10). In addition, anothernew peak which was not observed in the yeast cultures was detected. Thetotal ion chromatogram and mass spectrum of this new fatty acid wereconsistent with that of crepenynic acid (18:2_(Δ9(Z),12(c))) (FIGS. 4Band C), demonstrating that the CtFAD2-11 polypeptide had Δ12-acetylenaseactivity. As shown in Table 10, crepenynic acid accounted for 0.51% oftotal fatty acids.

It was observed that the expression of CtFAD2-11 transiently in the N.benthamiana cells resulted in a reduction in the content of the18:2^(Δ9(Z),12(Z)) relative to the untransformed control (Table 10).This was likely due to the competition of CtFAD2-11 with the endogenouscis-Δ12 oleate desaturase in the N. benthamiana cells for the availablepool of oleic acid, the substrate for both enzymes. Overall, the resultsfrom the yeast and N. benthamiana expression experiments indicated thatCtFAD2-11 functioned primarily as an oleate Δ12-desaturase lackingstereo-specificity, producing both linoleic acid and its trans-Δ12isomers. In addition, it could also further desaturate the Δ12 doublebond of linoleic acid to form the acetylenic bond of crepenynic acid.

The other ten candidate CtFAD2 polypeptides were also expressedtransiently in N. benthamiana leaves in the same manner, but we did notobserve any new fatty acids which were not present endogenously in N.benthamiana leaves, which already have high levels of FAD2.

TABLE 10 Fatty acid composition of N. benthamiana leaves transientlyexpressing CtFAD2-11. C16:0 C16:1 C16:2 C16:3 C18:0 C18:1 C18:1^(Δ11)Control 17.42 ± 0.48 0.25 ± 0.02 0.83 ± 0.12 7.24 ± 0.15 3.32 ± 0.331.02 ± 0.09 0.46 ± 0.03 CtFAD2-11 23.70 ± 2.57 0.28 ± 0.05 0.62 ± 0.095.50 ± 0.81 5.30 ± 0.72 3.82 ± 0.30 1.15 ± 0.36 C18:2^(Δ9Z, 12E) C18:2C18:3 C20:0 C20:1 C18:2Ac Control 12.03 ± 0.65 56.79 ± 0.19 0.46 ± 0.100.18 ± 0.15 CtFAD2-11 0.35 ± 0.07 11.63 ± 0.84 45.78 ± 4.01 0.95 ± 0.190.41 ± 0.04 0.51 ± 0.06 (n = 3)

DISCUSSION

The 11 candidate CtFAD2 genes described above that were identified insafflower represent the largest FAD2 gene family observed in any plantspecies that has been examined to date. Although only a single FAD2 genewas identified in Arabidopsis (Okuley et al., 1994), FAD2 appears to beencoded by multiple genes in most other plant genomes studied so far.Two distinct FAD2 genes have been described in soybean (Heppard et al.,1996), flax (Fofana et al., 2004; Khadake et al., 2009) and olive(Hernanze et al., 2005); three genes in sunflower (Martinez-Rivas etal., 2001) and Camelina sativa (Kang et al., 2011); and five genes incotton (Liu et al., 1998). In the amphitetraploid species Brassicanapus, 4-6 different FAD2 genes have been identified in each diploidsub-genome (Scheffler et al., 1997). All of the candidate CtFAD2 geneswere expressed in safflower plants, since the sequences were isolatedfrom cDNAs. This was examined further as described in Example 6.

Although comparable studies are lacking, it is clear that safflower isunusual with respect to FAD2 gene family evolution. Safflower is aself-pollinating diploid plant species which is most closely related toa wild diploid species Carthamus palaestinus and it is not known to haveextensive genome duplication or re-arrangement (Chapman and Burke,2007). The multiple FAD2 cDNAs that were identified could not beattributed to alternative splicing since the candidate FAD2 genes didnot contain introns in the coding region sequence. Rather, geneduplication was more likely responsible for creating the FAD2 familycomplexity in safflower. The topology of the phylogenetic tree showedthat gene duplications may have occurred at several hierarchical levels.For example, the CtFAD2-3, -4 and -5 polypeptides were more closelyrelated to the others in that clade than they were to other safflowerFAD2 sequences, indicating that more recent gene duplications may havebeen responsible for the emergence of this clade.

Example 6. Expression Level of FAD2 Candidate Genes in Safflower

Expression Profile of FAD2 Genes in Different Tissues

To determine tissue expression patterns of the various candidate CtFAD2genes, RT-PCR analyses were carried out as described in Example 1. TotalRNA was extracted from cotyledons, hypocotyls, root and leaf tissuesderived from safflower seedlings of 10 DAG of high linoleic genotype SU,and from flower tissues and developing embryo from flowering plants, andused in the assays. The oligonucleotide primers used for the analysesare listed in Table 11.

TABLE 11 Oligonucleotide primers used for RT-qPCR in the expressionprofile study of safflower FAD2 genes. Primer gene Sense sequenceAntisense sequence CtFAD2-1 5′-GTGTATGTCTGCCTCCGAGA -3′5′- GCAAGGTAGTAGAGGACGAAG -3′ (SEQ ID NO: 116) (SEQ ID NO: 117) CtFAD2-25′-GCCTCCAAAGATTCATTCAGGTC -3′ 5′- CAAGATGGATGCGATGGTAAGG -3′(SEQ ID NO: 118) (SEQ ID NO: 119) CtFAD2-3 5′-ACGTGGCGGTCTCAGGTT -3′5′- AGGCGGTGACAATTATGATATC -3′ (SEQ ID NO: 120) (SEQ ID NO: 121)CtFAD2-4 5′-AAGGCAGGCCGTGATGCCGAT -3′ 5′- AGTATTTGATCAATCCGCTGG -3′(SEQ ID NO: 122) (SEQ ID NO: 123) CtFAD2-5 5′-CAATACGGTAGAGGCCACACAG -3′5′- ATCATCTCTTCGGTAGGTTATG -3′ (SEQ ID NO: 124) (SEQ ID NO: 125)CtFAD2-6 5′-GACATGTGCTCACGTGGTGCAT -3′ 5′- GTTGCTAATATCCACACCCTA -3′(SEQ ID NO: 126) (SEQ ID NO: 127) CtFAD2-7 5′-CGAATCACACCCACGGGATC -3′5′- CTAAAGAATTTCCATGGTGTTAC -3′ (SEQ ID NO: 128) (SEQ ID NO: 129)CtFAD2-8 5′-GAGCAACGGAGAGAAGTAACC -3′ 5′- GAGGGATGATAGAAAGAGGTCC -3′(SEQ ID NO: 130) (SEQ ID NO: 131) CtFAD2-9 5′-CATGTGTGGCTGGAGGATTCGA -3′5′- GCACCGAGTTTAGCCTTTGTCT -3′ (SEQ ID NO: 132) (SEQ ID NO: 133)CtFAD2-10 5′-CCAACAAACAAACCATCTCTCG -3′ 5′- GAGAGACGGTGGAAGTAGGTG -3′(SEQ ID NO: 134) (SEQ ID NO: 135) CtFAD2-115′-CCATTGATCCACCCTTCACCTTA -3′ 5′- AAAGACATAGGCAACAACGAGATC -3′(SEQ ID NO: 136) (SEQ ID NO: 137) KASII 5′-CTGAACTGCAATTATCTAGG -3′5′- GGTATTGGTATTGGATGGGCG -3′ (SEQ ID NO: 138) (SEQ ID NO: 139)

The temporal and spatial expression pattern of the 11 CtFAD2 genes isshown in FIG. 5. The RT-qPCR assays showed that CtFAD2-1 was exclusivelyexpressed in developing seeds. In contrast, CtFAD2-2 was expressed atlow levels in seeds as well as other tissues examined. Further, noexpression of CtFAD2-4, -5, -6, -7, -8, -9 was observed in developingembryos. Low, yet detectable, levels of CtFAD2-10 and -11 expressionwere observed in developing seeds, more so in the late developmentalstage as the safflower seeds approach maturity. CtFAD2-4, -6, -7, -9 and-11 all showed high levels of expression in the young seedling tissuesincluding in cotyledons and hypocotyls. CtFAD2-5 and -8 appeared to beroot-specific and CtFAD2-10 was preferentially expressed in flowertissues, with relatively low levels detected in various other tissuesexamined, including developing seeds, and ten days old seedling tissues.

No amplification products were detected after 40 cycles of amplificationin control reactions with total RNA template but without reversetranscriptase, indicating the absence of contaminating genomic DNA inthe RNA preparations.

Example 7. Demonstration of the Genetic Mutation in the Safflower LineS317

The first identified high oleic trait in safflower, found in a safflowerintroduction from India, was controlled by a partially recessive alleledesignated ol at a single locus OL (Knowles and Hill, 1964). The oleicacid content of olol genotypes was usually 71-75% for greenhouse-grownplants (Knowles, 1989). Knowles (1968) incorporated the ol allele into asafflower breeding program and released the first high oleic (HO)safflower variety “UC-1” in 1966 in the US, which was followed by therelease of improved varieties “Oleic Leed” and the Saffola seriesincluding Saffola 317 (S-317), S-517 and S-518. The high oleic (olol)genotypes were relatively stable at different temperatures (Bartholomew,1971). In addition, Knowles (1972) also described a different allele ol₁at the same locus, which produced in homozygous condition between 35 and50% oleic acid. In contrast to olol genotype, the ol₁ol₁ genotype showeda strong response to temperature (Knowles, 1972).

Additional germplasm with higher oleic acid content (>85%) has beenreported (Fernandez-Martinez et al., 1993; Bergman et al., 2006). Oleiccontent up to 89% in safflower was reported by Fernandez-Martinez et al.(1993) in the germplasm accession PI401472 originally sourced fromBanglasesh. The Montola series developed by Bergman et al. (2006)contains more than 80% oleic acid, clearly beyond the uppermost level ofoleic acid in “UC-1” variety containing the olol allele as described byKnowles and Hill (1964). Genetic analysis through the crosses andsegregation analysis, the high oleic and very high oleic lines suggestedthat these two lines share the same alleles at the OL locus. The veryhigh oleic content (85%) was generated by the combination of the olalleles and modifying genes with a small positive effect on oleic acid(Hamdan et al., 2009).

In Vitro Biochemical Characterisation of the High Oleic Mutant LineS-317

Safflower microsomes were freshly prepared from developing seeds of thehigh oleic genotype S-317 at mid-maturity stage, about 15 days postanthesis (DPA), as described by Stymne and Appelqvist (1978). A standard90 μL reaction mixture contained 40 μg microsomal protein, 2 nmol[¹⁴C]oleoyl-CoA in 0.1 mmol potassium phosphate buffer pH7.2. Then, 10μL of 50 mM NADH was added and the incubation continued for anadditional 5, 10 or 20 min. The reactions were stopped by adding 90 μLof 0.15 M acetic acid and lipid extracted with 500 μL CHCl₃:MeOH (1:1).The lower CHCl3 phase was recovered and the polar lipids from itseparated by thin layer chromatography (TLC) using the solvent systemCHCl₃/MeOH/HAc/H₂O (90:15:10:3 v/v/v/v). Spots corresponding to PC werescraped off the plate and the associated fatty acyl groups weretransmethylated in 2 ml of 2% sulphuric acid in MeOH at 90° C. for 30min. The resultant FAMEs were separated on AgNO₃ treated TLC plates withhexane:DEE:HAc (85:15:1 v/v/v). ¹⁴C labelled oleate and linoleatemethylester standards were spotted on the plate as references. Theplates were exposed and analysed by a Fujifilm FLA-5000 phosphorimager.The radioactivity of each sample was quantified with Fujifilm MultiGauge software.

Upon the addition of NADH to the reactions with the wild-typemicrosomes, it was observed that the added [¹⁴C]oleoyl-CoA disappearedrapidly, within 10 min, at the same time as the appearance of[¹⁴C]linoleate, indicating the efficient conversion of oleate tolinoleate in the wild type safflower microsomes. In contrast, for thehigh oleic genotype S-317, a significantly higher ratio of [¹⁴C]oleateto [¹⁴C]linoleate was found in the in vitro reactions throughout thetime course (Table 12), indicating that the biosynthesis of linoleicacid via desaturation of oleate by the microsomes was drasticallyreduced in this genotype.

TABLE 12 Percentage of C18:2 product derived from C18:1 in safflowermicrosomes. Time WT HO (min) C18:1 C18:2 C18:1 C18:2 0 99.2 ± 1.1  0.8 ±1.1 100.0 ± 0.0  0.0 ± 0.0 5 79.6 ± 1.2 20.4 ± 1.2 99.4 ± 0.3 0.6 ± 0.310 69.6 ± 0.4 30.4 ± 0.4 95.4 ± 1.6 4.6 ± 1.6 20 60.6 ± 0.6 39.4 ± 0.695.1 ± 1.5 4.9 ± 1.5 n = 2Molecular Characterisation of the High Oleic Allele of

To understand the molecular basis of the high oleic genotype (olol) insafflower, the two seed-expressed FAD2 cDNAs, namely CtFAD2-1 andCtFAD2-2, were amplified by PCR from three high oleic varieties: S-317,LeSaf 496 and CW99-OL and sequenced. The cDNAs covering the entirecoding regions of the CtFAD2-1 genes from all three high oleic varietieswere identical in nucleotide sequence to each other, and shared about98% sequence identity with the CtFAD2-1 cDNA derived from the wild typevariety SU, including one nucleotide deletion and 22 nucleotidesubstitutions in the HO genotype relative to the wild-type. The singlebase pair deletion was found at nucleotide 606 counting from the firstATG, in approximately the middle of the CtFAD2-1 coding region. Thisdeletion caused a shift in the translational reading frame that createda stop codon soon after the deletion, so that the mutant gene in thethree olol varieties encoded a predicted, truncated polypeptide withoutthe third histidine box present in the wild-type protein (FIG. 6). Itwas noteworthy that there was a relatively high level of sequencevariation in the DNA sequences near the deleted single nucleotide siteof the ol allele, suggesting that additional mutations had accumulatedin the mutant gene.

The DNA regions including the 5′ UTR introns of CtFAD2-1 and CtFAD2-2were also isolated from the olol mutant S-317 and compared to thewild-type introns. The CtFAD2-1 intron from S-317 was 1144 bp in length,61 bp longer than the wild type SU intron which was 1083 bp in length.The comparison of the nucleotide sequences of the CtFAD2-1 intronsshowed an overall sequence identity of 76.8%, the introns differing in27 indels and 95 single nucleotide substitutions (FIG. 7).

Interestingly, the nucleotide substitutions in the mutant gene were notdistributed evenly throughout the 1142 bp long region corresponding tothe coding region of the defective CtFAD2-1, in that 14 of the 22(63.6%) substitutions were present near the nucleotide deletion, mostwithin 123 bp just downstream of the single nucleotide deletion. Incontrast, the CtFAD2-2 introns in the wild-type and mutant genotypesshared an overall 99.5% sequence identity, with only 12 nucleotidesubstitutions and one 2-nt indel. This indicates that either someselection pressure had occurred in the defective CtFAD2-1 gene in the HOmutant, or, perhaps more likely, that the CtFAD2-1 mutation was ofancient origin and might have originated from a progenitor species ofsafflower such as C. palaestinus.

An EMS mutant (S-901) derived from the commercial high oleic varietyS-518 has been described in U.S. Pat. No. 5,912,416. Although geneticstudies indicated that the so called ol₂ allele in this new genotype wasdistinct from the ol₁ and ol₁ alleles in the OL locus, its molecularnature was not determined by Weisker (U.S. Pat. No. 5,912,416). TheS-901 genotype was characterised by an increase of the level of oleicacid to 89.5-91.5% of total fatty acids in mature seeds. There was areduction of saturated fatty acids, i.e palmitic acid down to about 4%and stearic acid down to about 2.5%. However, S-901 did not display anormal plant phenotype and suffered some comprised growth and yield.Morphologically it was shorter and flower heads were smaller compared toits parent line S-518. It also flowered late and contained less oil inthe seeds.

Designing Perfect PCR Markers for High Oleic Breeding

The single nucleotide deletion-sequence polymorphism in the mutantCtFAD2-1 allele, concluded to be the causative mutation responsible forthe HO phenotype, was developed as the molecular basis of a highlyefficient molecular marker for tracking the mutant ol allele. Theinventors thus developed a molecular marker assay that allowed theidentification and selection of the mutant ol allele for breedingpurposes or varietal identification purposes, even when it was presentin the heterozygous state. Molecular marker assisted selection therebyeliminates the need to produce an extra generation of plants that mustbe screened for the fatty acid phenotype. Simple genetics combined withperfect molecular marker assays will make it possible for safflowerbreeders to quickly incorporate the high oleic trait in their breedingprogram.

It appeared that there was insufficient sequence variation in the exonsof CtFAD2-1 between the wild type SU and high oleic genotype S-317 toeasily generate a differential marker based on PCR reactions. However,the inventors could take advantage of the relatively high sequencedivergence in the 5′ UTR intron of CtFAD2-1 between the OL and olalleles. There were stretches of highly variable sequences between thesetwo alleles which enabled the design of unique PCR primers. Thefollowing illustrative primers were designed to amplify a specificproduct of 315 bp long from the high-oleic genotypes carrying the ololmutant allele, but not in the wild type SU. HO-Sense:5′-ATAAGGCTGTGTTCACGGGTTT-3′ (SEQ ID NO: 140); and HO-Antisense:5′-GCTCAGTTGGGGATACAAGGAT-3′ (SEQ ID NO: 141) (FIG. 7). Another pair ofillustrative primers specific for the wild-type gene in the variety SUwhich gave rise to a 603 bp PCR product as follows: HL-sense:5′-AGTTATGGTTCGATGATCGACG-3′ (SEQ ID NO: 142); and HL-antisense:5′-TTGCTATACATATTGAAGGCACT-3′ (SEQ ID NO: 143) (FIG. 7). A pair ofprimers derived from the safflower KASII gene, ctkasII-sense:5′-CTGAACTGCAATTATCTAGG-3′ (SEQ ID NO: 144) and ctkasII-antisense5′-GGTATTGGTATTGGATGGGCG-3′ (SEQ ID NO: 145) were used as the positivecontrol to ensure the equal loading and good PCR performance of thetemplate DNA.

The PCR reaction conditions were 94° C. for 2 min, followed by 40 cyclesof 94° C. for 30 sec, 58° C. for 30 sec and 72° C. for 30 sec. Thereaction products were separated by electrophoresis on a 1% agarose geland visualized under UV light following ethidium bromide staining of thegel. A fragment of about 300 bp was observed in the amplificationreactions for all five high oleic genotypes examined, namely S-317,5-517, CW99-OL, LeSaf496 and Ciano-OL, while such a fragment was absentfor the wild-type genotype SU. Conversely, a fragment of about 600 bpwas present in the amplifications for the wild-type safflower SU, butnot in any of the high oleic varieties tested. As a positive control, a198 bp band derived from KASII gene was amplified in the eractions forall of the tested lines. The amplicon's identities were verified by DNAsequencing.

The sequence divergence in the 5′UTR intron region of the CtFAD2-1 genebetween the high oleic and wild type safflower alleles therebyfacilitated the development of a PCR marker diagnostic for the presenceor absence of the CtFAD2-1 mutation. It was completely linked to the olallele whatever the genetic background, that is, it was a perfectlylinked marker. However, that molecular marker was a dominant marker andconsequently use of that marker alone would not allow the distinctionbetween homozygous and heterozygous genotypes for the ol allele. Toovercome this, another pair of PCR primers was designed which amplifiedonly the wild type Ol allele. Consequently, the use of such wild-typespecific primers in combination with high oleic specific PCR primersallowed the distinguishing between homozygous and heterozygous genotypesat the CtFAD2-1 locus.

CtFAD2-1 Expression is Drastically Reduced in High Oleic Genotypes

In above sections, it was shown that CtFAD2-1 was expressed only in thedeveloping embryos of the developing seeds and not detectably in variousother tissues examined, including in leaf, root, flower, cotyledon andhypocotyls derived from young safflower seedlings. CtFAD2-1 was highlyexpressed in developing seeds where the rate of fatty acid metabolismwas high, and led to active oil accumulation having mostly C18:2 in arelatively short period of time. CtFAD2-1 had its highest expressionlevel at about the mid point in seed development, with a more moderateexpression level at both early and late stages of seed development.

Using the RT-qPCR assay method, the level of normalized gene expressionof the CtFAD2-1 gene was measured in three high oleic varieties, namelyS-317, Lesaff496 and CW99-OL, and compared to that in developing seedsof the wild-type genotype SU. As can be seen in FIG. 8, CtFAD2-1expression was detected in all three stages of developing embryos in thewild-type safflower genotype SU, with the highest level of expressionobserved at the mid-maturity stage, consistent with the previous resultsand verifying the temporal transcription pattern for this key FAD2 gene.However, CtFAD2-1 transcripts were barely detectable in the three higholeic varieties S-317, Lesaff496 and CW99-OL (FIG. 8), indicating a highlevel of instability of the RNA transcripts from this gene in the mutantembryos.

In contrast, the levels of transcripts from CtFAD2-2 were similar forthe wild-type and high oleic genotypes, showing that CtFAD2-2 expressionwas unaffected in the HO embryos as well as demonstrating that the RNApreparations were suitably pure for the assays. Therefore, it wasconcluded that CtFAD2-2 expression could also contribute to theΔ12-desaturation of fatty acids for storage lipids in developingsafflower seeds, but at a much lower level than for CtFAD2-1 in thewild-type seeds, as well as being involved in Δ12-desaturation of fattyacids for membrane lipids in root, leaf and stem. There was no evidencethat CtFAD2-2 expression was elevated in the high oleic mutant inresponse to, or as compensation for, the loss of CtFAD2-1 activity inthe developing safflower seeds of the CtFAD2-1 mutant.

The Drastically Reduced CtFAD2-1 Transcripts in HO Lines are Caused byNon-Sense Mediated RNA Degradation (NMD)

The drastically reduced level of CtFAD2-1 transcripts in the HO embryosmight have been caused by non-sense mediated mRNA degradation (NMD) ofCtFAD2-1 mRNAs, since a premature stop codon was found in the middle ofcoding sequence soon after the single nucleotide deletion. The NMDsystem is considered to be a mechanism involved in the degradation ofaberrant mRNAs that contain a premature termination codon (PTC)resulting from unexpected errors such as genomic mutations,transcriptional errors, and mis-splicing. It is a mechanism that isuniversally present in eukaryotes and, in particular it has beenextensively studied in yeast and mammals. It is rather poorly studied inhigher plants, but there are a few reports including the soybean Kunitztrypsin inhibitor gene (Kti3), phytohemagglutinin gene (PHA) from commonbean (Jofuku et al., 1989; Voelker et al., 1990), pea ferredoxin gene(FED1) (Dickey et al., 1994) and rice waxy gene (Isshiki et al., 2001).

It was shown in these experiments that the ol mutation leading to thehigh oleic acid trait in safflower seedoil was correlated with lowlevels of CtFAD2-1 mRNA accumulation in the developing seeds. Previousresearch indicated that the ol allele was semi recessive, which was notconsistent with a posttranscriptional gene silencing mechanism mediatedby small RNAs. Gene silencing involves 21 to 24 nt siRNA produced fromdouble strand RNA, resulting from transcription of antisense or hairpinRNA and can act genetically as a dominant or semi dominant locus(Brodersen and Voinnet, 2006). To confirm that the mechanism of the olmutation was distinct from RNAi related gene silencing, we carried out asmall RNA sequencing, as follows.

Two small RNA libraries, derived from the high oleic genotype S-317 andwild type SU, were generated using pooled RNA isolated from themid-maturity developing embryos. Bulk sequencing of the small RNAlibraries was performed with Solexa technology (Hafner et al., 2008).Sequencing of these two libraries was performed on the Illumina's SolexaSequencer and the samples were run side by side. The sequencing of SUand S-317 small RNA libraries generated a total of 23,160,261 and21,696,852 raw reads, respectively. Analysis of these reads resulted inidentification of 22,860,098 and 21,427,392 sequences ranging in lengthfrom 18 to 30 nucleotides (nt), respectively. The presence anddistribution of small RNA corresponding to CtFAD2-1 in the SU and S-317libraries were determined. Only low, barely detectable levels of smallRNAs corresponding to CtFAD2-1 were detected from both small RNAlibraries and distributed almost evenly over the coding regions ofCtFAD2-1 genes. There was no clear difference between the wild type andhigh oleic libraries.

From this data, it was concluded that small-RNA mediatedpost-transcriptional gene silencing was not the main mechanism by whichthe accumulation of the mutant CtFAD2-1 transcripts was prevented.

Transient Expression Studies in N. benthamiana Leaves

To investigate the NMD phenomenon further, the inventors performedexperiments for the transient expression of CtFAD2-1 derived from bothwild-type and high oleic genotypes in N. benthamiana leaves.

Each of the CtFAD2-1 ORFs was inserted in sense orientation into amodified pORE04 binary vector under the control of the CaMV-35Spromoter. Agrobacterium tumefaciens strain AGL1 harbouring either the35S:CtFAD2-1 or its mutant form 35S:CtFAD2-1Δ was infiltrated into theunderside of the fully expanded leaves of N. benthamiana together with35S:P19, as described in Example 5. Following a period of 5 days furthergrowth at 24° C., the infiltrated regions were excised and total RNAswere obtained from the samples using an RNeasy Mini Kit (Qiagen). Tomeasure the CtFAD2-1 RNA levels, Real Time qPCR assays were carried outin triplicate using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen)and run on ABI 7900HT Sequence Detection System as described inExample 1. PCR was carried out under the following conditions: aninitial 48° C. for 30 min, then 95° C. for 10 min, followed by 40 cyclesof 95° C. for 15 s and 60° C. for 60 s. The primers for the exogenousCtFAD2-1 gene were: sense: 5′-GTGTATGTCTGCCTCCGAGA-3′ (SEQ ID NO: 146);antisense: 5′-GCAAGGTAGTAGAGGACGAAG-3′ (SEQ ID NO: 147). A referencegene, safflower CtKASII, was used to normalize the expression levels;its specific primers were: sense: 5′-CTGAACTGCAATTATCTAGG-3′ (SEQ ID NO:144); and antisense: 5′-GGTATTGGTATTGGATGGGCG-3′ (SEQ ID NO: 145). Highlevels of CtFAD2-1 expression were observed in the N. benthamiana leavesfrom the 35S-CtFAD2-1 gene derived from the wild-type SU variety. Incomparison, much lower levels of expression were observed for the35S-CtFAD2-1Δ gene derived from the high oleic genotype.

A. thaliana ecotype Col-0 plants were transformed with A. tumefaciensstrain AGL1 carrying a binary vector harbouring a seed-specific promoterFp1 driving either the CtFAD2-1 or the CtFAD2-14 coding region,according to the method of Clough and Bent (1998). Total RNA wasisolated from siliques containing mid-maturity stage embryos of progenyof the resultant transformed plants using an RNeasy Mini Kit (Qiagen).Gene expression studies were done using the RNA preparations by the RealTime RT-qPCR assays, carried out in triplicate as described above. Highlevels of CtFAD2-1 expression were observed in the Arabidopsis siliquesexpressing the Fp1-CtFAD2-1 derived from SU, however, the expression ofFp1-CtFAD2-1Δ derived from the high oleic genotype was drasticallyreduced in comparison.

It was demonstrated that CtFAD2-1 specific small RNAs were not producedat significantly higher levels in developing high oleic safflower seedscompared to small RNAs from the wild-type gene, even though the mutantCtFAD2-1 transcript was drastically reduced in amount. It was thereforeconcluded that the reduction in CtFAD2-1 RNA in the high oleic genotypewas due to NMD, distinct from a small RNA mediated posttranscriptionalgene silencing mechanism. The NMD phenomenon was also observed when themutant coding region was expressed exogenously in either the N.benthamiana leaves or the Arabidopsis siliques.

Example 8. Isolation of Safflower cDNAs which are Candidates forEncoding FATB

Isolation of Safflower FATB cDNA Sequences

Safflower seed oil contains approximately 7% palmitic acid. This fattyacid is synthesized in the plastids of the developing seed cells, fromwhere it is exported to the cytosol of the cells for its incorporationinto triacylglycerols. The key enzyme for palmitic acid export ispalmitoyl-ACP thioesterase which hydrolyses the thioester bond betweenthe palmitoyl moiety and the acyl carrier protein (ACP) to which theacyl group is covalently bound while it is synthesised in the plastid.The enzyme palmitoyl-ACP thioesterase belongs to a group of solubleplastid-targeted enzymes designated

FATB. In seedoil plants, this enzyme displays specificity towards shortchain saturated acyl-ACP as substrate. A gene encoding FATB enzyme wasinitially isolated from plant species accumulating medium chain-lengthsaturated fatty acids, such as lauric acid (C12:0) from California baytree (Umbellularia californica). Subsequent studies demonstrated thatFATB orthologues were present in all plant tissues, predominantly inseeds, with substrate specificity ranging from C8:0-ACP to C18:0-ACP. InArabidopsis and most temperate oilseed crops including safflower,palmitic acid is the major saturated fatty acid in seed oil.

To isolate safflower cDNAs that encoded candidates for FATB, the cDNAlibrary of developing safflower seeds was screened using a heterologousprobe consisting of a FATB cDNA fragment from cotton (Gossypiumhirsutum) as described in Example 1. One full length cDNA, namedCtFATB-T12, was isolated from safflower seed cDNA library. This cDNAcontained an open reading frame of 1029 nucleotides in length, encodinga polypeptide of 343 amino acids. Its 5′ and 3′ UTRs were 236 nt and 336nt in length, respectively. It was predicted that the CtFATB-T12polypeptide had a predicted transit peptide of about 60 amino acids anda 210-amino acid residue core that contained two repeats of a helix andmulti-stranded sheet fold common to the so-called hot dog fold proteins.

From the Compositae Genome Project (CGP) expressed sequence tag (EST)database for safflower (cgpdb.ucdavis.edu/cgpdb2), three different ESTswere identified with homology to CtFATB-T12, namely EL379517, EL389827,and EL396749. Each was partial length. The corresponding genes weredesignated CtFATB-A, CtFATB-B and CtFATB-C, respectively. The fulllength cDNA CtFATB-T12 isolated from the safflower seed cDNA library wasidentical in nucleotide sequence to the EST from CtFATB-C in theiroverlapping region. It appeared that CtFATB-A was more divergent in itsnucleotide sequence in comparison to the other two CtFATB sequences.

Expression Profile of CtFATB Genes by Real-Time qPCR Analysis

The gene expression profile of the three CtFATB genes was studied withReal time qPCR as outlined in Example 1. Oligonucleotide primerscorresponding to the unique region of each of the three genes weredesigned, including CtFATB-A, sense primer:5′-AGAGATCATTGGAGACTAGAGTG-3′ (SEQ ID NO: 148); antisense primer:5′-CCCATCAAGCACAATTCTTCTTAG-3′ (SEQ ID NO: 149); CtFATB-B, sense primer:5′-CTACACAATCGGACTCTGGTGCT-3′ (SEQ ID NO: 150); antisense primer:5′-GCCATCCATGACACCTATTCTA-3′ (SEQ ID NO: 151); CtFATB-C, sense primer:5′-CCTCACTCTGGGACCAAGAAAT-3′ (SEQ ID NO: 152); antisense primer:5′-TTCTTGGGACATGTGACGTAGAA-3′ (SEQ ID NO: 153). PCR reactions performedin triplicate as described in Example 1.

As shown in FIG. 9, CtFATB-A showed low expression levels in leaves,roots and in all three stages of developing embryos that were examined.CtFATB-B was active in leaves and roots, but showed lower expression indeveloping embryos than in leaves and roots. This suggested that thisgene might play only a minor role, if any, in fatty acid biosynthesis indeveloping seeds. In contrast, CtFATB-C demonstrated high expressionlevels across all the tissues examined, particularly in the developingembryos. This indicated that CtFATB-C was the key gene encoding FATB forthe production of palmitic acid in safflower seed oil. This wasconsistent with our recovery of only one FATB cDNA clone from the seedembryo library, namely CtFATB-T12 which was identical in sequence toCtFATB-C. Based on these data, an approximately 300 bp DNA fragmentderived from CtFATB-T12 (CtFATB-C) was chosen as the gene sequence to beused in the preparation of hpRNA constructs for down-regulation of FATBin safflower seed, as described in the following Examples.

Example 9: Isolation and Expression of Safflower cDNA Encoding FAD6

Isolation of Safflower FAD6 cDNA Sequence

The distinct fatty acid compositions found in microsomal andchloroplastic membrane lipids and seed storage oils are the result of anintricate metabolic network that operates to control this composition byregulating fatty acid biosynthesis and flux through both the so-calledprokaryotic and eukaryotic pathways. It is clear that microsomal FAD2enzyme has a major role in converting oleate to linoleate in the ERfollowing export of oleic acid from the plastid and conversion to CoAesters in the cytoplasm. Chloroplast omega-6 desaturase (FAD6) is anenzyme that desaturates 16:1 and 18:1 fatty acids to 16:2 and 18:2,respectively, on all 16:1- or 18:1-containing chloroplast membranelipids including phosphatidyl glycerol, monogalactosyldiacylglycerol,digalactosyldiaclyglycerol, and sulfoguinovosyldiacylglycerol. AnArabidopsis fad6 mutant was reported to be deficient in desaturation of16:1 and 18:1 to 16:2 and 18:2, respectively, on all chloroplast lipids(Browse et al., 1989). When the fad6 mutant was grown at low temperature(5° C.), the leaves become chlorotic and the growth rate wassignificantly reduced compared to the wild-type (Hugly and Somerville,1992). A cDNA sequence encode FAD6 was first isolated from Arabidopsisby Falcone et al. (1994). Since then, cDNAs encoding FAD6 and FAD6 geneshave been isolated from several plant species including Brassica napus,Portulaca oleracea, soybean, and Ricinus communis.

In order to isolate a cDNA clone encoding the chloroplast ω6 desaturaseencoded by the FAD6 gene from safflower, the CPG database was searchedfor homologous sequences. Eight EST sequences, namely EL378905,EL380564, EL383438, EL385474, EL389341, EL392036, EL393518, EL411275were identified and assembled into a single contig sequence of 808 nt.This sequence had an intact 5'end but was incomplete at 3'-end. The fulllength cDNA was subsequently obtained through 3' RACE PCR amplificationusing, as template, DNA extracted from a lambda cDNA library made fromdeveloping seeds of safflower (SU). PCR conditions were as described inExample 2. A single oligo primer, designated ctFAD6-s2 was used in theamplification reaction in combination with M13 Forward primer since thesequence for this primer was present in the vector of cDNA library. Thesequence of the ctFAD6-s2 primer was: 5'-CATTGAAGTCGGTATTGATATCTG-3'(SEQ ID NO: 154). A cDNA of 1545 bp was obtained which had an openreading frame of 1305 bp that encoded a candidate FAD6 polypeptide of435 amino acids. This polypeptide shared between 60-74% amino acidsequence identity with other cloned plant FAD6 polypeptides. Adendrogram showing the phylogenetic relationship between the safflowerFAD6 sequence and representative FAD6 plastidial Δ12 desaturaseidentified in higher plants was generated by Vector NTI (FIG. 10).

Expression Profile of CtFAD6 by Real-Time qPCR Analysis

The expression profile of CtFAD6 was studied by Real time RT-qPCR whichwas carried out using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen)and run on an ABI 7900HT Sequence Detection System with defaultsparameters as described in Example 1. The primers used were: ctFAD6-S2:5′-CATTGAAGTCGGTATTGATATCTG-3′ (SEQ ID NO: 155) and ctFAD6-a2:5′-GTTCCAACAATATCTTCCACCAGT-3′ (SEQ ID NO: 156). Reactions wereperformed in triplicate in 10 μL total volumes containing 20 ng of totalRNA template, 800 mM each primer, 0.25 μL of reverse transcriptase and 5μL one-step RT-PCR master mix reagents. Conditions for RT andamplification were 48° C. for 30 min, then 95° C. for 10 min, followedby 40 cycles of 95° C. for 15 s and 60° C. for 60 s. Expression of areference gene safflower CtkasII was used to normalize the FAD6expression levels. The calculations were made as described in Example 1.

The analysis showed that CtFAD6 was expressed at relatively low levelsin leaves, roots and three consecutive stages of developing embryos. Thelow expression levels observed in developing seeds was consistent withthe notion that FAD6 might have a relatively minor role in thedesaturation of oleate in seeds.

Example 10. Design and Preparation of Genetic Constructs to SilenceFatty Acid Biosynthesis Genes in Safflower

Hairpin RNAs (hpRNA) are a type of RNA molecule that have been usedextensively to reduce gene expression in plants. Hairpin RNAs aretypically transcribed in plant cells from a DNA construct containing aninverted repeat of a sequence derived from a gene to be silenced. ThehpRNA transcript thereby has complementary sense and antisense sequenceswhich hybridise to form a double-stranded RNA (dsRNA) region joined by aloop sequence. Such dsRNA structures are processed by endogenoussilencing machineries in the plant cells to form small RNA molecules ofabout 21 to 24 nucleotides corresponding in sequence to the gene to bereduced in activity. These small RNAs can form complexes with endogenousproteins that specifically silence the gene of interest. Such silencingcan occur at the transcriptional level, mediated by DNA methylation ofparts of the target gene, at the post-transcriptional level bydegradation of the target mRNA, or by binding to the mRNAs to inhibitits translation and thereby reduce protein synthesis encoded by thegene. When the hpRNA includes a sequence that is in common betweenmembers of a gene family, the hpRNA can silence each of those genes thathave the sequence.

Safflower has a large family of FAD2 genes, with at least 11 membersidentified as described herein (Examples 2 to 6). Safflower also hasmultiple genes encoding FATB polypeptides, with at least three membersidentified (Example 8), and at least one FAD6 gene (Example 9). Indeed,the experiments described above may not have identified all members ofthe gene families in safflower. To determine which members of the FAD2and FATB gene families were involved in the biosynthesis of linoleicacid or of saturated fatty acids found in safflower seed oil,particularly palmitic acid, and whether FAD6 also was, several geneticconstructs were made to express hpRNA molecules in safflower seed, tosilence various combinations of FAD2, FATB and FAD6 genes. Each of thesehpRNA constructs was designed to be expressed specifically in developingsafflower seeds during the period of oil synthesis. This was done byusing either foreign promoters or promoters isolated from safflower toexpress the constructs, which were introduced into safflower by planttransformation.

Construction of pCW600

A plant binary expression vector was designed for the expression oftransgenes in seeds using the promoter of an Arabidopsis Olesoin1 gene(TAIR website gene annotation At4g25140) (SEQ ID NO: 52). The isolatedpromoter was 1192 bp in length starting from nucleotide 12899298 inAccession No. NC003075.7, except that within the 1198 bp sequence, 6 bpwere omitted to avoid common restriction digestion sequences to aidlater cloning steps. The AtOleosin promoter has previously been used forstrong, seed-specific expression of transgenes in safflower and Brassicaspecies (Nykiforuk et al., 1995; Vanrooijen and Moloney, 1995). Thispromoter was likely to be a bi-directional promoter, directing strongseed-specific expression of coding regions joined to both ends of thepromoter fragment. The Arabidopsis oleosin promoter shares features withthe Brassica napus oleosin promoter, characterised to have abi-functional nature (Sadanandom et al., 1996). The promoter waschemically synthesised, cloned into pGEMT-Easy and the EcoRI fragmentcontaining the promoter blunted via the Klenow fragment enzyme fill-inreaction, and ligated into the Klenow-blunted HindIII site of pCW265(Belide et al., 2011), generating pCW600 (AtOleosinP::empty). Thisvector had a selectable marker gene that encoded a hygromycinphosphotransferase (HPT), thereby allowing selection for tolerance tohygomycin in tissue culture during the transformation process. Thevector also included a 35S::GFP gene which allowed selection oftransformed cells or tissues by fluorescence under UV lightillumination. By inserting the AtOleosin promoter, the vector wasdesigned for expression of a coding region of interest which could beinserted into a multiple cloning site situated downstream of thepromoter and upstream of a nos polyadenylation signal (nos3′). Thisvector served as the backbone vector for the constructs pCW602 andpCW603 described below.

Construction of pXZP410

A flax linin promoter (U.S. Pat. No. 7,642,346) (SEQ ID NO: 53) wasinserted as a NotI-XhoI fragment into the binary vector pT7-HELLSGATE12(Wesley et al., 2001), generating the Gateway silencing binary vectorpXZP410. The vector pXZP410 had a selectable marker gene which conferredresistance to kanamycin during tissue culture and allowing seed specificexpression of hairpin RNA constructs under the control of the lininpromoter. The vector had two introns, one in the sense orientation andthe other in the antisense direction with respect to the promoter, andhad AttL1 and AttL2 recombinational sites flanking the introns.

To make a silencing construct from pXZP410, two copies of a sequencefrom the target gene in the Gateway entry vector to be silenced wereinserted into the vector, one inserted 5′ and the other 3′ of the twointrons, in inverted orientation with respect to each other to form aninverted repeat. The recombinational sites readily allowed insertion ofthe two copies by using Gateway recombinase cloning systems (Invitrogen,Carlsbad, USA), as described previously (Wesley et al., 2001). Thisvector pXZP410 was used as a backbone vector for producing pCW631 andpCW632 as described below.

Construction of pCW571

A 300 bp sequence (SEQ ID NO: 50) identical to a region of the safflowergene CtFATB-3 corresponding to nucleotides 485-784 of the CtFATB-3 cDNAwas chemically synthesised and inserted into pENTR/D topo (Invitrogen)according to the manufacturer's instructions, generating a Gateway entryclone designated pCW569. A 756 bp fragment (SEQ ID NO: 49) of the cDNAfor CtFAD2-2, corresponding to nucleotides 427-1182, was synthesized andamplified by RT-PCR from RNA isolated from developing safflower seeds.The primers were D28-PstI-5 (5′-CCTGCAGGTACCAATGGCTCGACGACACTG-3′) (SEQID NO: 157) and D28-AscI-3 (5′-CGGCGCGCCTTCACCTCCTCATCTTTATCC-3′) (SEQID NO: 158) respectively. The primers included 5′ PstI and 3′ AscIrestriction enzyme sites, thereby allowing the insertion of theamplified fragment into the corresponding sites of pCW569, generatingpCW570. This vector contained the fused regions from the CtFATB andCtFAD2-2 genes as a fragment of 1080 bp flanked by recombinationalsites, AttL1 and AttL2. Two copies of this FATB-FAD2-2 fragment werethen inserted into pXZP410, the second copy inverted with respect to thefirst, using LR clonase according to the supplier's instructions(Invitrogen, Carlsbad, USA). The resultant plasmid pCW571 had the flaxlinin promoter to transcribe the inverted repeat region in aseed-specific manner, in order to produce a hpRNA for reducingexpression of the CtFATB and CtFAD2-2 genes in seeds.

Construction of pCW603

The DNA fragment from pCW571 containing the inverted repeat of theCtFATB-CtFAD2-2 fragments with the two intervening introns was cut outwith SpeI, blunted using the Klenow I fragment of DNA polymerase andligated into the EcoRV site of pCW600, generating pCW603. This constructpCW603 was capable of expressing an hpRNA under the control of theAtOleosin promoter in seeds of safflower, to reduce expression of CtFATBand CtFAD2-2.

Construction of pCW581

A 590 bp fragment (SEQ ID NO: 51) of DNA made up of a 290 bp fragment ofCtFAD6, corresponding to nucleotides 451 to 750 of the cDNA for CtFAD6,and a 300 bp fragment of CtFATB, as for pCW571, was chemicallysynthesised and inserted into pENTR/D topo, generating pCW579. A 780 bpfragment of CtFAD2-2, as described above for pCW570, was cloned into theAscI site of pCW579, generating pCW580. This construct was an entryclone vector containing the sequences from the CtFATB, CtFAD6 andCtFAD2-2 genes joined in that order as a DNA fragment of 1370 bp withflanking recombinational sites, AttL1 and AttL2. Two copies of thisFATB-FAD6-FAD2-2 fragment were then inserted as an inverted repeat intopXZP410 using LR clonase, generating pCW581. This construct pCW581 was abinary vector having a flax linin promoter operably linked to theinverted repeat, which upon transcription in developing safflower seedscells was capable of expressing an hpRNA to reduce expression of theCtFATB, CtFAD6 and CtFAD2-2 genes.

Construction of pCW602

The DNA fragment containing the inverted repeat of the joinedCtFATB-CtFAD6-CtFAD2-2 regions, with the two intervening introns, wasenzymatically cut out of pCW571 with NotI, blunted using Klenow Ifragment and then ligated into the EcoRV site of pCW600, generatingpCW602. pCW602 had the CtFATB-CtFAD6-CtFAD2-2 sequences under thecontrol of the AtOleosin promoter, in contrast to pCW581 which had thesame design and gene fragments except with the linin promoter.

Construction of pCW631 and pCW632

Although the linin promoter was useful for expressing hpRNA in seeds,both pCW571 and pCW581 had the selectable marker that conferredkanamycin tolerance. In preliminary safflower transformationexperiments, we observed that the explants were not sufficientlysusceptible to kanamycin. Therefore, the kanamycin resistance cassetteof pCW571 and pCW581 was replaced with a hygromycin resistance cassetteas the selectable marker gene. The hygromycin resistance gene made up ofthe enCUP promoter:hygromycin:nos3'polyadenylation region was cut out ofpCW265 with SpeI-AvrII restriction digestion and used to replace thekanamycin resistance cassette in pCW571 and pCW581, thus generatingpCW631 and 632, respectively.

In summary, the constructs used in this first set of safflowertransformations contained the following main elements:

Vector Promoter Gene fragments in the inverted repeat pCW631 lininCtFAD2-2 and CtFATB pCW632 linin CtFAD2-2, CtFAD6 and CtFATB pCW602AtOleosin CtFAD2-2, CtFAD6 and CtFATB pCW603 AtOleosin CtFAD2-2 andCtFATB

These constructs were introduced into Agrobacterium strain AGL1 and usedto transform safflower as described in Example 1, with the results asfollows.

Example 11. Transformation of Safflower with Gene Silencing Constructs

The genetic constructs were used to transform excised cotyledons andhypocotyls of safflower variety S317 using the Agrobacterium-mediatedmethod with rescue of regenerated shoots using grafting (Belide et al.,2011). Over 30 independent transformed shoots growing on non-transformedroot-stocks (hereinafter termed T₀ plants) were regenerated for thevector pCW603 and grown to maturity as described in Example 1.Integration of the T-DNAs in the T₀ safflower scions was checked by PCRusing T-DNA vector-specific primers as described by Belide et al.(2011). Most plants found to be lacking the T-DNA used in the particulartransformation, presumed to be “escapes” from the hygromycin selectionduring regeneration in tissue culture, were discarded. However, somewere maintained as ‘null’ plants or negative controls for comparisonwith the transformed plants. These control plants were treated under thesame conditions as the transformed material in tissue culture, graftingand glass house conditions.

Example 12. Analysis of the Fatty Acid Composition of Seedoil ofTransgenic Safflower

Fatty acid analyses were conducted on individual T₁ seeds obtained fromthe transformed safflower plants, as follows. 30 independent T₀ plantstransformed with pCW603 in the S317 genetic background were grown in thegreenhouse and self-fertilised to produce seed. As many as 10 matureseed from a single seedhead from each T₀ plant were analysed for thelipid composition using GC analysis as described in Example 1. Resultsof the fatty acid composition analysis from seeds of safflower S317transformed with pCW603 are summarised in Table 13. As each transformedT₀ safflower plant was expected to be heterozygous for the T-DNA andtherefore produce a segregating population of Ti seeds, it was expectedthat the analysis of 5-10 seeds from each plant would include some null(segregant) seeds. Such null segregant seeds were good negative controlsin this experiment as they had grown and developed within the sameseedhead as the transformed seeds from the same plant. As can be seenfrom the data in Table 13, levels of oleic acid above 87% (as a weight %of the total fatty acid content) were observed in 6 independent lines(Lines 9, 12, 14, 20, 34 and 36) of the 30 lines generated. Many of thetransformed seeds had oleic acid contents in the range 87-91.7%, withlinoleic acid levels of 2.15 to 5.9% and palmitic acid levels of2.32-3.45%. The levels of other fatty acids in the seeds were notsignificantly different to the untransformed controls. The maximum oleicacid content observed in the Ti safflower seed transformed with pCW603was 91.7%, compared to approximately 77% in the non-transformed S317control seeds and the null segregant seeds. Notably, the seed lipidswere also significantly reduced in the levels of 16:0, decreasing from4.5% down to as low as 2.3%. The fatty acid profiles of the TAGfractions of the seedoils as purified on TLC plates were notsignificantly different to that of the total lipid extracted from theseeds.

Two metrics were calculated based on the total fatty acid composition ofthe safflower seeds, accounting for the most important fatty acids inthe seedoil. These were the oleic acid desaturation proportion (ODP) andthe palmitic+linoleic to oleic proportion (PLO). These were calculatedfor each seed and the data is shown in Table 13. Wild-type seeds (S317,untransformed) and the null segregants had an ODP ratio of about 0.1500and a PLO value of about 0.2830. The seeds transformed with the T-DNAfrom pCW603 exhibited significant reductions in the ODP and PLO values.13 seed generated from 6 independent events had a PLO value of less than0.1 and an ODP less than 0.06. One transformed line had an ODP of 0.0229and a PLO of 0.0514.

Mature individual single seeds of one elite line, S317 transformed withpCW603, line 9, and the untransformed parent S317 were subjected toLC-MS lipidomics analysis. These analyses clearly showed that the oilfrom the seeds transformed with the AtOleosinp:CtFATB-CtFAD2-2 RNAihairpin construct had dramatically altered TAG and DAG compositions(FIGS. 11 and 12). There was a clear increase in the level of TAG (54:3)and a decrease of TAG (54:5), which were predominantly18:1/18:1/18:1-TAG (triolein) and 18:1/18:2/18:2-TAG, respectively.Among the seeds analysed by LC-MS, the triolein (=54:3) content in TAGwas as much as 64.6% (mol %) at the highest oleic acid level (>90%,Table 14) for seed from the RNAi silencing line, compared to theuntransformed (S-317) parent which had triolein levels ranging between47% to 53%. The second most abundant oleate-containing TAG was18:0/18:1/18:1, followed by 18:1/18:1/18:2. The clearest differencebetween these safflower oils could also be seen in the DAG lipid class.The DAG(36:1) level was doubled in the seedoil from the transformed seedcompared to the parent seed, while DAG(36:4) was reduced by up to 10%.DAG(36.1) is predominantly 18:0/18:1-DAG and DAG(36:4) is 18:2/18:2-DAG(di-linoleate). The DAGs in the seed lipids were only a minor component,as the levels of total TAGs were about 100 times higher than total DAGs(Table 15).

Growth and Morphology of the Transgenic Plants

The T₀ safflower transformants containing the T-DNA from pCW603generated T₁ seed that segregated for the T-DNA yielding a set ofhomozygotes, hemizygotes and null segregants. The ratio of thesesub-populations depended on the number and linkage of T-DNA insertionevents in the T₀ plants, as was expected according to Mendeliangenetics. Therefore Ti seeds from each T₀ plant were analysedindividually. Analysis of the lipid profiles from individual seedsclearly showed that a single seed heads contained both null andtransgenic events.

TABLE 13 Lipid fatty acid composition of individual safflower T1 seedstransformed with the T-DNA of pCW603 in the S- 317 background. The levelof each fatty acid (%) was expressed as a percentage of the total fattyacid content. Sample* C16:0 C18:0 C18:1 C18:1d11 C18:2 C18:3 C20:0 C20:1PLO** ODP*** S317 (1) 4.60 1.47 75.69 0.69 16.49 0.00 0.31 0.26 0.278670.1789 S317 (2) 4.64 1.47 77.02 0.68 15.09 0.00 0.32 0.28 0.25620 0.1638S317 (3) 4.56 1.38 76.20 0.68 16.14 0.00 0.32 0.26 0.27161 0.1748 S317(4) 4.61 1.57 76.41 0.69 15.64 0.00 0.34 0.26 0.26506 0.1699 S317 (5)4.55 1.51 77.90 0.69 14.28 0.00 0.33 0.25 0.24176 0.1549 Null (1) 4.772.10 78.07 0.85 13.82 0.00 0.38 0.00 0.23815 0.1504 Null (2) 4.93 1.9676.02 0.89 15.55 0.00 0.38 0.28 0.26942 0.1698 Null (3) 5.59 2.22 75.150.92 15.68 0.00 0.45 0.00 0.28302 0.1726 Null (4) 4.61 1.65 78.52 0.7813.58 0.00 0.35 0.28 0.23163 0.1474 Null (5) 5.57 2.93 78.10 0.93 11.960.00 0.51 0.00 0.22445 0.1328 TS603.12 (5) 2.56 2.05 91.73 0.84 2.150.00 0.37 0.29 0.05136 0.0229 TS603.09 (4) 2.32 1.87 91.45 0.74 3.090.00 0.20 0.34 0.05911 0.0326 TS603.09 (5) 2.66 2.43 91.41 0.78 2.450.00 0.27 0.00 0.05587 0.0261 TS603.09 (1) 2.42 2.08 91.17 0.74 3.020.00 0.23 0.33 0.05972 0.0321 TS603.36 (1) 2.65 2.40 91.01 0.67 2.250.00 0.45 0.32 0.05379 0.0241 TS603.36 (2) 2.73 1.77 90.53 0.69 3.360.00 0.36 0.32 0.06728 0.0358 TS603.20 (4) 2.94 1.31 89.63 0.88 4.580.00 0.31 0.34 0.08393 0.0486 TS603.34 (4) 3.21 2.55 89.62 0.89 2.920.00 0.48 0.32 0.06847 0.0316 TS603.14 (2) 2.99 1.74 89.31 0.88 4.390.00 0.35 0.34 0.08257 0.0468 TS603.09 (3) 2.90 1.75 89.00 0.83 5.520.00 0.00 0.00 0.09465 0.0584 TS603.34 (5) 3.36 2.23 88.92 0.85 3.890.00 0.43 0.32 0.08151 0.0419 TS603.34 (2) 3.24 1.76 88.74 1.02 4.510.00 0.37 0.37 0.08725 0.0483 TS603.14 (1) 3.21 1.51 88.65 0.88 5.050.00 0.34 0.37 0.09310 0.0539 TS603.20 (3) 3.29 1.39 88.44 0.98 5.900.00 0.00 0.00 0.10391 0.0625 TS603.12 (2) 3.00 1.63 88.42 0.75 5.600.00 0.31 0.30 0.09722 0.0595 TS603.20 (2) 3.45 1.66 88.36 0.99 5.540.00 0.00 0.00 0.10175 0.0590 TS603.20 (1) 3.30 1.46 88.16 0.89 5.530.00 0.33 0.31 0.10021 0.0590 TS603.14 (5) 3.45 1.66 87.43 0.87 5.880.00 0.36 0.35 0.10673 0.0630 TS603.14 (4) 3.37 1.84 87.36 0.88 5.810.00 0.39 0.35 0.10509 0.0624 TS603.36 (4) 3.24 2.21 87.33 0.67 5.600.00 0.42 0.30 0.10117 0.0602 TS603.36 (3) 3.42 2.33 86.82 0.71 5.680.00 0.45 0.32 0.10491 0.0614 TS603.14 (3) 3.58 1.61 86.50 0.86 6.740.00 0.36 0.35 0.11931 0.0723 TS603.12 (4) 3.68 1.39 85.46 0.92 7.970.00 0.29 0.30 0.13624 0.0853 TS603.12 (3) 3.55 2.06 85.01 0.74 8.020.00 0.35 0.27 0.13609 0.0862 TS603.23 (5) 4.93 1.75 82.63 1.04 8.750.00 0.41 0.31 0.16561 0.0958 TS603.17 (5) 4.68 1.57 82.06 0.70 10.370.00 0.33 0.31 0.18339 0.1122 TS603.17 (4) 4.41 1.31 81.94 0.68 11.110.00 0.28 0.28 0.18940 0.1194 TS603.24 (3) 4.24 2.17 81.70 0.68 10.280.00 0.42 0.27 0.17772 0.1118 TS603.24 (5) 4.48 2.18 81.15 0.69 10.560.00 0.41 0.27 0.18541 0.1152 TS603.24 (4) 4.39 2.16 80.94 0.70 10.880.00 0.41 0.25 0.18869 0.1185 TS603.23 (4) 4.38 2.01 80.86 0.82 11.230.00 0.40 0.29 0.19312 0.1220 TS603.24 (2) 4.50 2.39 80.70 0.68 10.790.00 0.44 0.26 0.18946 0.1180 TS603.17 (3) 4.60 1.75 80.65 0.68 11.690.00 0.35 0.27 0.20203 0.1266 TS603.24 (1) 4.28 2.01 80.54 0.70 11.530.00 0.40 0.27 0.19632 0.1252 TS603.17 (2) 4.40 1.75 80.33 0.73 11.920.00 0.35 0.28 0.20316 0.1292 TS603.06 (3) 4.38 1.60 80.22 0.88 12.310.00 0.34 0.28 0.20803 0.1330 TS603.06 (5) 4.52 1.47 80.11 0.84 12.500.00 0.29 0.27 0.21245 0.1350 TS603.15 (3) 4.65 2.01 79.94 0.85 11.920.00 0.38 0.26 0.20720 0.1297 TS603.34 (3) 4.77 2.44 79.79 0.86 11.420.00 0.45 0.28 0.20281 0.1252 TS603.36 (5) 4.92 2.43 79.78 0.68 11.240.00 0.44 0.26 0.20258 0.1235 TS603.06 (1) 4.49 1.89 79.53 0.82 12.610.00 0.38 0.29 0.21495 0.1368 TS603.23 (2) 4.47 1.74 79.45 0.86 12.860.00 0.35 0.28 0.21810 0.1393 TS603.28 (4) 4.99 2.08 79.45 0.94 12.140.00 0.41 0.00 0.21554 0.1325 TS603.28 (1) 4.73 2.31 79.42 0.85 12.260.00 0.43 0.00 0.21390 0.1337 TS603.28 (5) 5.04 1.96 79.37 0.97 12.660.00 0.00 0.00 0.22301 0.1376 TS603.28 (2) 4.95 2.16 79.33 0.88 12.290.00 0.39 0.00 0.21728 0.1341 TS603.15 (2) 4.55 1.73 79.31 0.89 12.900.00 0.36 0.27 0.21995 0.1399 TS603.06 (4) 4.60 1.73 79.25 0.88 12.930.00 0.33 0.28 0.22121 0.1403 TS603.15 (1) 4.50 2.11 79.11 0.84 12.790.00 0.39 0.26 0.21860 0.1392 TS603.12 (1) 4.28 1.33 79.08 0.76 13.820.21 0.27 0.26 0.22880 0.1507 TS603.06 (2) 4.63 2.24 79.08 0.78 12.580.00 0.42 0.27 0.21765 0.1373 TS603.09 (2) 4.39 1.72 78.80 0.73 13.300.19 0.34 0.30 0.22453 0.1462 TS603.23 (1) 4.56 2.11 78.63 0.79 13.240.00 0.39 0.27 0.22648 0.1442 TS603.17 (1) 4.43 1.49 78.47 0.74 13.920.37 0.31 0.26 0.23386 0.1540 TS603.15 (4) 4.67 1.99 78.41 0.84 13.480.00 0.36 0.26 0.23138 0.1467 TS603.28 (3) 4.89 1.67 78.31 0.88 13.290.31 0.35 0.31 0.23208 0.1479 TS603.34 (1) 4.65 2.13 77.75 0.83 13.580.35 0.41 0.30 0.23441 0.1519 TS603.23 (3) 4.59 1.70 77.28 0.85 14.630.31 0.36 0.28 0.24866 0.1620 TS603.15 (5) 4.69 1.64 77.23 0.97 14.850.00 0.35 0.27 0.25302 0.1613 *samples labelled with this convention:TS603.12(5) denotes the plant transformed with vector pCW603, event 12,seed 5. Samples labelled as ‘null’ are determined via PCR analysis asnon-transformed escapes in the plant transformation. **PLO metriccalculated as (16:0 + 18:2)/18:1 ***ODP metric calculated as (18:2 +18:3)/(18:1 + 18:2 + 18:3)

TABLE 14 Fatty acid composition in single seed total lipid (% of totalfatty acids). Sample C16:0 C18:0 C18:1 C18:1d11 C18:2 C18:3 C20:0 C20:1S317 Seed 1 5.21 2.35 77.36 0.88 14.20 0.00 0.00 0.00 S317 Seed 2 5.083.04 77.20 0.80 13.88 0.00 0.00 0.00 S317 Seed 3 5.00 2.65 78.89 0.7812.67 0.00 0.00 0.00 TS603.9 Seed 1 3.76 3.07 87.93 0.91 4.34 0.00 0.000.00 TS603.9 Seed 2 3.33 2.98 89.97 0.93 2.80 0.00 0.00 0.00 TS603.9Seed 4 3.61 4.28 88.20 0.87 3.05 0.00 0.00 0.00 TS603.9 Seed 5 3.61 3.1388.45 0.92 3.38 0.00 0.50 0.00 TS603.9 Seed 6 4.45 3.04 85.50 0.90 5.620.00 0.51 0.00

TABLE 15 Relative TAG and DAG amount. Sample TAG/DAG ratio S317 Seed 160.4 S317 Seed 2 84.7 S317 Seed 3 71.2 TS603.9 Seed 1 117.5 TS603.9 Seed2 124.2 TS603.9 Seed 4 97.8 TS603.9 Seed 5 96.5 TS603.9 Seed 6 95.9

Seeds from the some of the transgenic lines were grown under controlledconditions (temperature, soil, optimal watering and fertilising, butunder natural lighting) in the greenhouse to observe the plantmorphology and growth rate. No phenotypic differences were observedbetween the transformed T₁ plants and their null segregant siblings.Transformed seeds germinated at the same rate as the untransformed seedsand yielded seedlings having the same early seedling growth rate(vigour). All of the sown seeds became established and grew into fullyfertile plants. DNA was prepared from tips of true leaves fromindividual plants of the Ti generation and PCR analysis was conducted todetermine the ratio of null and transgenic plants. As expected, nullsegregants were identified.

This phenotypic analysis indicated that safflower plants transformedwith the T-DNA of pCW603 and expressing the transgenes did not sufferany detrimental effects compared to null segregants.

The safflower seeds of the T2 generation were tested for oilcomposition. Seed from several plants having high levels of oleic acid(Table 13) were grown into mature plants producing second generationseed (T2 seed). These seed were harvested when mature and analysed forthe fatty acid composition of their oil. The data are given in Table 16.Although the T1 seed displayed up to about 92% oleic acid, the T2 seedreached 94.6% oleic acid. The observed increase in the T2 generationrelative to the T1 generation may have been due to homozygosity of thetransgene, or simply to the large number of lines analysed.

Southern blot hybridisation analysis is used to determine the number ofT-DNA insertions in each transformed line, and lines with a single T-DNAinsertion are selected. The oil content of T2 seeds is not significantlydifferent to that in the control, untransformed seeds of the samegenetic background and grown under the same conditions.

Analysis of Safflower Seeds Transformed with the T-DNA from pCW631

The T1 seed of safflower variety S-317 transformed with pCW631 weresimilarly analysed for their fatty acid composition. Table 17 shows thedata and the ODP and PLO metrics for these seeds. The oleic acid contentin lipid of these seeds was up to 94.19%. The palmitic acid content ofseed TS631-01 T1 (21) having the highest level of oleic acid was 2.43%,the ODP was 0.0203 and the PLO was 0.0423. These analyses demonstratedthat the hairpin RNA construct in pCW631 generally produced higher oleicacid levels that the construct in pCW603 when transformed into safflowerof the S-317 genetic background. This observation indicated that thelinin promoter used in pCW631 expressed the hairpin RNA more strongly orwith a better timing of expression, or a combination of both, relativeto the AtOleosin promoter used in pCW603.

The T2 seeds of safflower variety S-317 transformed with the T-DNA frompCW631 were analysed by GC. Levels of oleic acid up to 94.95% wereobserved with an ODP of 0.01 and PLO of 0.035.

Safflower seeds and plants transformed with the T-DNAs from theconstructs pCW632 and pCW602 are analysed in the same manner as for theseed and plants transformed with pCW603 and pCW631. GC analysis of theT1 seed of safflower variety S-317 transformed with pCW632 showed oleicacid level of up to 94.88%, with ODP as 0.0102 and PLO as 0.0362. TheirT2 seeds showed up to 93.14% oleic acid, with ODP as 0.0164 and PLO as0.0452.

Extraction of Larger Volumes of Safflower Seed Oil

T4 seeds from the homozygous transgenic line designated TS603-22.6 wereharvested and total seedoil extracted using the Soxhlet apparatus asdescribed in Example 1. Aliquots of extracted oil were analysed by GC(Table 18). A total of 643 grams oil was recovered. Extractions 2, 3, 5,and 6 were pooled, while extractions 4, 7 and 8 were pooled in aseparate lot. The mixtures were further analysed for fatty acidcomposition by GC. The data are shown in Table 18.

Example 13. Design and Preparation of Further Gene Silencing Constructs

Based on the results described in Examples 10-12, other gene silencingconstructs were prepared as follows to increase the oleic acid contentof safflower seed oil and decrease the ODP or PLO ratios. These genesilencing constructs included combining different promoters, fromnon-safflower sources as well as safflower sources, to achieve maximalreduction in the safflower FAD2-2, FATB-3 and FAD6 gene expression, andfurther silencing more than one FAD2 gene in addition to FAD2-2. Theseconstructs are used to transform varieties of safflower which haveinactivated versions of the endogenous CtFAD2-1 gene, such as S-317,Ciano-OL and Lesaff496.

Construction of pCW700

This plant binary expression vector has two foreign (non-safflower)promoters with different but overlapping expression patterns insafflower seed, rather than one promoter, to produce hairpin RNA toreduce expression of the endogenous CtFATB and CtFAD2-2. The twopromoters are the AtOleosin promoter and the flax linin promoter and thetwo hpRNA expression cassettes are in the same T-DNA molecule. Thisvector is constructed by restriction digestion of the hpRNA geneexpression cassette from pCW631, having the linin promoter and hpRNAencoding region for silencing of CtFAD2-2 and CtFATB, and inserted itinto the T-DNA of pCW603, thus generating a construct encoding a hairpinRNA against these two safflower genes. This construct is used totransform safflower varieties such as Lesaff496, Ciano-OL and S-317.

TABLE 16 Fatty acid composition of lipid from individual safflower T2seeds transformed with the T-DNA of pCW603 in the S-317 background. Thelevel of each fatty acid (%) was expressed as a percentage of the totalfatty acid content. Sample C16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 ODPPLO TS603-22.3 T2 (4) 2.4 0.9 94.6 1.7 0.0 0.2 0.0 0.0181 0.043956TS603-22.6(5) 2.5 1.0 94.5 2.0 0.0 0.0 0.0 0.0214 0.048069 TS603-22.6(2)2.1 0.9 94.4 2.5 0.0 0.0 0.0 0.0266 0.04934 TS603-22.6(4) 2.4 1.0 94.32.3 0.0 0.0 0.0 0.0239 0.049028 TS603-22.4 T2 (2) 2.2 1.5 94.3 1.8 0.00.0 0.1 0.0200 0.042739 TS603-22.6 T2 (20) 2.2 1.1 94.2 2.4 0.0 0.0 0.00.0256 0.048622 TS603-22.05(4) T2 2.2 1.4 94.2 2.0 0.0 0.1 0.0 0.02120.044211 TS603-22.8 T2 (1) 2.1 1.1 93.9 2.5 0.2 0.1 0.0 0.0263 0.04839TS603-22.6 T2 (10) 2.8 1.0 93.9 2.1 0.0 0.0 0.0 0.0223 0.052359TS603-22.6 T2 (6) 2.9 1.1 93.9 2.0 0.0 0.0 0.0 0.0214 0.052318TS603-22.05(1) T2 2.2 1.3 93.8 2.2 0.1 0.2 0.0 0.0240 0.04783 TS603-22.6T2 (13) 2.9 1.2 93.8 1.7 0.0 0.2 0.0 0.0185 0.049564 TS603-22.6 T2 (16)2.9 0.9 93.8 2.3 0.0 0.0 0.0 0.0249 0.055517 TS603-22.4 T2 (1) 2.5 1.593.7 1.9 0.1 0.1 0.0 0.0204 0.046933 TS603-22.6(1) 2.7 1.2 93.7 2.3 0.00.0 0.0 0.0241 0.052497 TS603-22.3 T2 (5) 2.5 1.1 93.6 2.6 0.0 0.0 0.00.0278 0.05449 TS603-08.2(4) 2.2 0.8 93.6 3.3 0.0 0.0 0.0 0.0355 0.05861TS603-22.6 T2 (9) 2.9 1.2 93.5 2.2 0.0 0.2 0.0 0.0235 0.054815TS603-22.6 T2 (19) 2.9 1.1 93.5 2.3 0.0 0.0 0.0 0.0245 0.055483TS603-22.4 T2 (3) 2.4 1.0 93.5 2.8 0.1 0.0 0.0 0.0299 0.056044TS603-22.1 T2 (2) 2.7 0.9 93.5 2.9 0.0 0.0 0.0 0.0306 0.059281TS603-22.6 T2 (14) 3.1 1.5 93.4 2.1 0.0 0.0 0.0 0.0222 0.054943TS603-08.2(3) 2.7 0.9 93.4 2.9 0.0 0.0 0.0 0.0306 0.059851TS603-22.05(5) T2 2.3 1.6 93.4 2.5 0.2 0.0 0.0 0.0267 0.051234TS603-22.6 T2 (15) 3.0 1.2 93.3 2.4 0.0 0.0 0.0 0.0261 0.057723TS603-22.5 T2 (11) 2.2 1.2 93.3 2.3 0.0 0.3 0.4 0.0285 0.048123TS603-22.6 T2 (8) 2.9 1.2 93.3 2.5 0.0 0.0 0.0 0.0265 0.057514TS603-22.5 T2 (12) 2.2 1.3 93.3 2.3 0.0 0.3 0.4 0.0289 0.047835TS603-22.6 T2 (7) 3.1 1.3 93.2 2.5 0.0 0.0 0.0 0.0265 0.059228TS603-22.4 T2 (8) 2.1 1.8 93.2 2.0 0.0 0.3 0.3 0.0254 0.044186TS603-22.6 T2 (11) 2.9 1.2 93.1 2.5 0.0 0.1 0.0 0.0271 0.057755TS603-22.3 T2 (1) 2.5 1.2 93.1 3.1 0.0 0.0 0.0 0.0335 0.060295TS603-22.5 T2 (14) 2.1 1.3 93.1 2.6 0.0 0.2 0.4 0.0318 0.050656TS603-22.5 T2 (16) 2.3 1.5 93.0 2.3 0.0 0.3 0.3 0.0283 0.049621TS603-22.5 T2 (18) 2.3 1.0 93.0 2.8 0.0 0.2 0.4 0.0341 0.05407TS603-22.5 T2 (15) 2.0 0.9 92.9 3.0 0.0 0.3 0.5 0.0381 0.054149TS603-22.6 T2 (12) 3.0 1.2 92.9 2.7 0.0 0.0 0.0 0.0294 0.061518TS603-22.05(3) T2 2.5 1.0 92.9 3.3 0.1 0.2 0.0 0.0350 0.061372TS603-22.6 T2 (18) 2.9 0.8 92.9 3.2 0.0 0.0 0.0 0.0349 0.06593TS603-22.6(3) 2.7 0.7 92.9 3.6 0.0 0.0 0.0 0.0383 0.067112 TS603-22.8 T2(4) 2.6 1.2 92.9 3.3 0.0 0.0 0.0 0.0358 0.063416 TS603-22.6 T2 (17) 2.70.8 92.9 3.5 0.0 0.0 0.0 0.0375 0.067028 TS603-22.1 T2 (1) 2.7 1.1 92.83.1 0.2 0.0 0.0 0.0334 0.062312 TS603-22.4 T2 (11) 2.2 1.4 92.6 2.7 0.00.3 0.4 0.0340 0.053189 TS603-22.4 T2 (12) 2.1 1.4 92.6 2.9 0.0 0.3 0.40.0355 0.054077 TS603-22.1 T2 (4) 2.4 0.9 92.6 4.0 0.0 0.0 0.0 0.04340.069653 TS603-44(2) T1 2.7 1.1 92.5 3.3 0.0 0.2 0.0 0.0358 0.065506TS603-22.8 T2 (5) 2.9 1.1 92.4 3.5 0.0 0.0 0.0 0.0381 0.069194TS603-22.5 T2 (6) 2.1 2.0 92.4 2.4 0.0 0.3 0.4 0.0305 0.049301TS603-22.4 T2 (5) 2.8 1.7 92.4 2.9 0.0 0.0 0.0 0.0318 0.062593TS603-22.4 T2 (13) 2.3 1.1 92.3 3.3 0.0 0.2 0.4 0.0406 0.060311TS603-22.3 T2 (3) 2.7 1.4 92.3 3.4 0.1 0.1 0.0 0.0368 0.065979TS603-34.3 T2 (1) 2.6 1.1 92.3 3.9 0.0 0.1 0.0 0.0421 0.070162TS603-34.3 T2 (13) 2.4 1.7 92.2 2.7 0.0 0.3 0.3 0.0332 0.055508TS603-22.5 T2 (10) 2.4 1.6 92.1 3.0 0.0 0.3 0.3 0.0366 0.058947TS603-19.02(3) T2 2.5 2.0 92.1 3.2 0.0 0.1 0.0 0.0345 0.061861TS603-08.2(1) 2.6 1.0 92.1 4.1 0.0 0.0 0.0 0.0444 0.073162 TS603-22.1 T2(3) 2.5 1.1 92.1 4.0 0.1 0.2 0.0 0.0435 0.070895 TS603-22.5 T2 (8) 2.21.6 92.1 3.1 0.0 0.3 0.4 0.0381 0.058128 TS603-19.2 T2 (11) 2.4 0.9 91.93.9 0.0 0.2 0.4 0.0463 0.067902 TS603-44(1) T1 3.0 0.9 91.9 4.0 0.0 0.10.0 0.0439 0.076059 TS603-19.02(1) T2 2.4 1.8 91.7 3.4 0.2 0.2 0.10.0384 0.063629 TS603-22.3 T2 (2) 3.0 1.1 91.7 4.0 0.0 0.0 0.0 0.04340.076459 TS603-22.5 T2 (7) 2.3 1.7 91.7 3.2 0.0 0.3 0.4 0.0392 0.060643TS603-22.4 T2 (9) 2.3 1.3 91.7 3.6 0.0 0.3 0.5 0.0442 0.064255TS603-22.5 T2 (9) 2.4 1.6 91.6 3.4 0.0 0.3 0.4 0.0414 0.063679TS603-22.4 T2 (7) 2.4 1.3 91.5 3.9 0.0 0.3 0.4 0.0462 0.068489TS603-22.4 T2 (10) 2.4 1.4 91.5 3.7 0.0 0.3 0.5 0.0453 0.066056TS603-34.3 T2 (2) 2.7 1.6 91.4 3.9 0.0 0.1 0.1 0.0442 0.072984TS603-22.4 T2 (4) 2.7 1.3 91.4 4.2 0.1 0.1 0.0 0.0461 0.076004TS603-19.02(5) T2 2.4 1.9 91.4 4.1 0.1 0.2 0.0 0.0445 0.070234TS603-10.02(2) T2 2.7 1.5 91.2 4.4 0.1 0.1 0.0 0.0480 0.077276TS603-22.5 T2 (13) 2.4 1.5 91.2 4.1 0.0 0.2 0.4 0.0489 0.071172TS603-19.02(4) 2.6 1.8 91.1 4.3 0.2 0.1 0.0 0.0471 0.075467 TS603-22.4T2 (6) 2.4 1.6 91.0 4.1 0.0 0.3 0.4 0.0488 0.07045 TS603-34.4 T2 (2) 2.71.7 90.8 4.4 0.1 0.1 0.0 0.0487 0.078563 TS603-27.5 T2 (1) 2.7 1.3 90.74.3 0.0 0.3 0.3 0.0511 0.076932 TS603-19.02(2) T2 2.7 1.7 90.6 4.8 0.00.2 0.0 0.0532 0.083056 TS603-22.5 T2 (17) 2.7 1.7 90.5 4.0 0.0 0.4 0.40.0480 0.074387 TS603-08.2(5) 3.0 1.1 90.5 5.3 0.0 0.0 0.0 0.05830.091395 TS603-19.2 T2 (17) 2.6 1.8 90.5 4.2 0.0 0.3 0.3 0.0504 0.074894TS603-19.2 T2 (10) 2.5 2.2 90.4 3.9 0.0 0.4 0.3 0.0464 0.070963TS603-34.3 T2 (3) 2.8 1.5 90.2 5.2 0.1 0.1 0.0 0.0575 0.088097TS603-19.2 T2 (19) 2.5 2.3 90.2 4.0 0.0 0.3 0.3 0.0481 0.073009TS603-09.8 T2 (1) 2.8 1.3 90.0 5.0 0.0 0.3 0.3 0.0589 0.086178 S317 (1)5.57 2.93 78.10 11.96 0.00 0.51 0.00 0.1531 0.224455 S317 (2) 4.77 2.1078.07 13.82 0.00 0.38 0.00 0.1770 0.23815 S317 (3) 4.55 1.51 77.90 14.280.00 0.33 0.25 0.1865 0.241763 S317 (4) 4.61 1.65 78.52 13.58 0.00 0.350.28 0.1764 0.231634

TABLE 17 Lipid fatty acid composition analysis of individual safflowerT2 seeds transformed with the T-DNA of pCW631 in the S-317 background.The level of each fatty acid (%) was expressed as a percentage of thetotal fatty acid content. FAME ID# Sample C16:0 C16:1 C18:0 C18:1 C18:2C20:0 C20:1 C22:0 ODP PLO 916 TS631-01 T1 (21) 2.43 0.13 1.06 94.19 1.560.24 0.39 0.27 0.0203 0.0423 912 TS631-01 T1 (17) 2.37 0.10 1.17 93.731.87 0.23 0.34 0.19 0.0230 0.0452 913 TS631-01 T1 (18) 2.57 0.11 0.9793.27 2.56 0.22 0.38 0.19 0.0305 0.0550 897 TS631-01 T1 (16) 2.50 0.101.04 93.24 2.35 0.21 0.37 0.18 0.0247 0.0521 910 TS631-03 T1 (1) 2.420.11 1.60 93.00 2.01 0.30 0.34 0.23 0.0250 0.0476 921 TS631-04 T1 (1)2.49 0.11 1.31 92.93 2.34 0.27 0.34 0.20 0.0275 0.0521 895 TS631-01 T1(14) 2.54 0.11 1.07 92.82 2.56 0.25 0.37 0.20 0.0316 0.0549 917 TS631-01T1 (22) 2.66 0.17 1.16 92.75 2.68 0.28 0.42 0.30 0.0334 0.0576 918TS631-03 T1 (2) 2.36 0.11 2.02 92.64 1.92 0.36 0.32 0.25 0.0242 0.0463914 TS631-01 T1 (19) 2.68 0.12 1.23 92.30 2.84 0.28 0.34 0.21 0.03450.0598 898 TS631-02 T1 (1) 2.99 0.12 1.21 90.79 4.06 0.26 0.32 0.260.0483 0.0777 915 TS631-01 T1 (20) 3.74 0.00 1.07 90.67 4.14 0.00 0.000.38 0.0456 0.0869 919 TS631-03 T1 (3) 4.23 0.10 1.50 81.70 11.92 0.300.29 0.27 0.1494 0.1976 899 TS631-02 T1 (2) 4.54 0.08 1.61 80.87 12.070.31 0.26 0.25 0.1525 0.2054 900 TS631-02 T1 (3) 4.47 0.08 2.07 80.6411.90 0.37 0.24 0.23 0.1506 0.2030 901 TS631-02 T1 (4) 4.60 0.09 1.8380.24 12.51 0.29 0.22 0.22 0.1586 0.2132 906 TS631-02 T1 (9) 4.44 0.091.74 80.20 12.76 0.31 0.23 0.24 0.1619 0.2144 903 TS631-02 T1 (6) 4.470.10 1.60 79.99 13.06 0.30 0.25 0.24 0.1664 0.2191 907 TS631-02 T1 (10)4.39 0.09 1.82 79.90 12.98 0.33 0.24 0.24 0.1655 0.2174 909 TS631-02 T1(12) 4.31 0.09 1.52 79.87 13.48 0.27 0.26 0.19 0.1720 0.2227 905TS631-02 T1 (8) 4.46 0.09 1.57 79.58 13.47 0.30 0.30 0.23 0.1730 0.2254902 TS631-02 T1 (5) 4.72 0.08 1.45 79.05 13.87 0.32 0.27 0.24 0.17890.2351

TABLE 18 Soxhlet Extraction of oil and fatty acid profile of oil.Extraction Dry seed Meal Recovered Oil Fatty acid composition (Wt %) Noweight (g) weight (g) oil (g) content (%) 16:0 16:1 18:0 18:1 18:2 18:320:0 20:1 22:0 Ext-1 223.00 219.89 68.14 30.99 2.7 0.1 1.3 92.7 2.6 0.00.3 0.3 0.1 Ext-2 233.24 232.71 64.68 27.79 2.8 0.1 1.4 91.9 3.0 0.0 0.30.3 0.1 Ext-3 240.07 238.72 72.37 30.32 3.1 0.1 1.5 91.9 2.8 0.0 0.3 0.30.1 Ext-4 231.59 229.96 66.69 29.00 3.0 0.1 1.5 91.4 3.6 0.0 0.3 0.3 0.1Ext-5 220.22 219.54 62.64 28.53 2.9 0.1 1.3 92.1 3.0 0.0 0.3 0.3 0.1Ext-6 288.97 288.20 77.75 26.98 2.7 0.1 1.5 92.3 2.8 0.0 0.3 0.3 0.1Ext-7 241.10 239.73 71.96 30.02 2.9 0.1 1.4 91.9 3.2 0.0 0.3 0.2 0.1Ext-8 243.68 242.90 67.04 27.60 2.9 0.1 1.6 91.4 3.4 0.0 0.3 0.3 0.1Ext-9 321.26 320.76 92.15 28.73 3.3 0.1 1.6 89.6 4.8 0.0 0.3 0.3 0.1Mixtures Ext-2/3/5/6 2.9 0.1 1.4 92.0 2.9 0.0 0.3 0.3 0.2 Ext-4/7/8 2.90.1 1.5 91.3 3.4 0.0 0.3 0.3 0.2Construction of pCW701-pCW710

Safflower-derived promoters, expected to have optimal activity insafflower seeds, are isolated using DNA sequencing technologies thatprovide accurate sequence information for the regions of DNA upstreamand downstream of an expressed gene. Previous results, as described inExamples 2 to 6, have shown that the CtFAD2-1 gene is highly expressedduring seed development in safflower. Therefore, the promoter region ofthis gene is an excellent candidate for driving efficient transgeneexpression in safflower seeds. As shown in Example 6, CtFAD2-2 wasactive in genetic backgrounds where CtFAD2-1 was inactivated bymutation. Therefore the promoter of CtFAD2-2 is used in safflower todrive expression of hairpin RNAs targeting CtFAD2-2 activity, amongstother genes. Other promoter elements useful for expression of transgenesin safflower seeds include endogenous (i.e. safflower) promoter elementsin the upstream parts of genes for Oleosin (CtOleosin) and seed-storageproteins such as 2S and 11S proteins (Ct2S and Ct11S). The promoterelements of CtFAD2-1, CtOleosin, Ct2S and Ct11S are isolated usingstandard PCR-based techniques based on safflower genome sequences, andincorporated into plant binary expression vectors. These promoterelements are used to express hpRNA silencing molecules in the constructspCW701-pCW710 or in conjunction with other non-safflower promotersexpressing the same or different hpRNA genes such as in pCW602, pCW603,pCW631 or pCW632. Combinations of hpRNA genes with different promotersare also produced by crossing transformed plants with the individualgenes, typically where the hpRNA genes are unlinked.

To isolate the CtFAD2-1 promoter, a genomic DNA fragment of about 3000bp upstream of the CtFAD2-1 translation start ATG codon is isolatedusing PCR-based techniques and used to replace the AtOleosin promoterfrom pCW603 and pCW602, thus generating the constructs pCW701 andpCW702, respectively.

To isolate the CtFAD2-2 promoter, a genomic DNA fragment of about 3000bp upstream of the CtFAD2-2 translation start ATG codon is isolatedusing PCR-based techniques and used to replace the AtOleosin promoterfrom pCW603 and pCW602, thus generating the constructs pCW703 andpCW704, respectively.

To isolate the CtOleosin-1 promoter, a genomic DNA fragment of about1500 base pairs upstream of the CtOleosin translation start ATG codon isisolated using PCR-based techniques and used to replace the AtOleosinpromoter from pCW603 and pCW602, thus generating the constructs pCW705and pCW706, respectively.

To isolate the Ct2S promoter, a genomic DNA fragment of about 1500 basepairs upstream of the Ct2S translation start ATG codon is isolated andused to replace the AtOleosin promoter from pCW603 and pCW602, thusgenerating the vectors pCW707 and pCW708, respectively.

To isolate the Ct11S promoter, a genomic DNA fragment of about 1500 basepairs upstream of the Ct11S start ATG is isolated from genomic DNA ofsafflower using PCR-based techniques and used to replace the AtOleosinpromoter from pCW603 and pCW602, thus generating the vectors pCW709 andpCW710, respectively.

Each of these vectors is transformed into safflower varieties asdescribed in Example 1.

Example 14. Field Performance of Safflower Varieties

A series of non-transformed varieties and accessions of safflower weregrown in the summer of 2011-2012 at a field station located at Narrabri,New South Wales. Seeds were sown within 5 m×3 m field plots into heavyclay soil commonly found in the Narrabri region. Plants were exposed tonatural light and rainfall except that they were irrigated once after 4weeks of growth. Mature seed were harvested and samples of about 50seeds were analysed for lipid content and fatty acid composition inseedoil. The oleic acid contents in seedoil of the various varieties andaccessions are shown in Table 19 and FIG. 13.

The data from the field trial indicated that there was a range of oleicacid contents of the safflower seed, surprising in the extent of theobserved range. Most notably, various accessions described as ‘higholeic’ and previously reported to provide seedoil with at least 70%oleic acid, such Ciano-OL, only produced about 42-46% oleic acid.Linoleic acid levels were much higher than expected based on previousreports. In contrast, other accessions that were reported to give higholeic contents did indeed produce high oleic acid levels (60%-76%) inseedoils under field conditions, such as accessions PI-5601698 andPI-560169. The reason for the considerably lower oleic acid levels thanexpected in some accessions was believed to be related to the presenceof CtFAD2-1 alleles other than the ol allele, such as for example, theol1 allele which is temperature sensitive, and to growing conditionsthat were less than ideal in the 2011-12 season. Further fatty acidanalysis on the seed obtained from field grown safflower will be carriedout to confirm the variation observed in the oleic acid content of someaccessions.

TABLE 19 Lipid fatty acid composition of safflower varieties grown inthe field. ID C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 PI 6134635.3 <0.1 5.5 9.1 78.8 0.1 0.6 0.2 PI 537677 6.3 0.2 2.3 10.3 79.9 0.10.3 0.2 PI 537645 7.8 0.2 2.2 10.5 78.0 0.1 0.4 0.2 PI 572433 6.4 0.12.5 10.7 78.7 0.2 0.4 0.2 PI 572472 6.3 0.2 2.4 11.1 78.7 0.2 0.4 0.2 RC1002 L 6.8 0.2 2.7 11.5 77.3 0.2 0.4 0.2 PI 537701 6.7 0.2 2.3 11.6 77.80.2 0.4 0.2 CC1485-3-1-1-1-1 7.8 0.2 2.2 11.6 76.9 0.2 0.4 0.2 PI 5601637.0 0.2 1.9 11.7 77.8 0.1 0.4 0.2 PI 451958 7.1 0.2 2.7 11.8 77.1 <0.10.4 0.2 PI 306609 6.6 0.2 2.5 12.0 77.3 0.2 0.4 0.2 PI 451950 6.6 0.12.2 12.4 77.5 0.1 0.4 0.2 PI 537705 6.6 0.2 2.6 13.1 76.3 0.2 0.4 0.2 PI413718 7.1 0.2 2.9 13.4 74.9 0.2 0.6 0.2 PI 560161 7.1 0.2 2.1 14.4 75.00.1 0.4 0.2 PI 560180 6.5 0.2 2.0 18.3 71.5 0.3 0.4 0.2 PI 401477 5.80.2 2.3 36.8 53.2 0.2 0.5 0.2 PI 537712 6.5 0.2 2.5 40.5 48.5 0.2 0.50.2 PI 537695 6.0 0.2 2.3 41.9 47.9 0.2 0.5 0.3 CIANO-OL 6.9 0.3 1.942.9 46.5 0.2 0.5 0.3 PI 538779 6.6 0.2 2.1 43.2 46.2 0.2 0.4 0.3Sinonaria 6.0 0.2 2.1 43.5 46.6 0.2 0.4 0.2 PI 401479 5.9 0.2 2.1 48.241.7 0.3 0.5 0.4 PI 560166 5.7 0.2 2.3 55.0 35.0 0.1 0.5 0.3 PI 4014745.5 0.3 1.9 59.3 30.6 0.4 0.6 0.6 PI 560177 5.1 0.2 1.9 60.7 30.3 0.20.4 0.3 PI 603207 5.6 0.2 2.0 65.2 25.0 0.2 0.5 0.3 PI 560167 5.8 0.21.6 65.9 24.8 0.2 0.4 0.3 PI 560174 5.9 0.3 1.8 67.6 22.6 0.3 0.5 0.3 PI603208 5.4 0.2 2.3 68.3 21.7 0.2 0.6 0.3 PI 538025 5.3 0.2 2.3 68.5 21.60.3 0.4 0.3 PI 560168 5.9 0.2 1.8 68.5 21.8 0.3 0.5 0.3 PI 560169 5.60.2 1.9 71.8 18.6 0.2 0.5 0.3 PI 577808 5.3 0.2 2.0 75.0 15.5 0.3 0.50.3 PI 612967 5.4 0.2 1.8 76.3 14.5 0.2 0.4 0.3

This experiment also showed that the level of oleic acid in seedoilobtained from plants grown in the field was typically about 5-10% lowerthan from plants grown in the greenhouse, even for the best performedaccessions in the field. The reason for this was thought to be thatfield growing conditions were less ideal than in the greenhouse.

In a further experiment, plants of safflower cultivar S317 were grown ineither the field or the greenhouse to compare the fatty acid compositionof seedoil. Even though the field conditions were more favourable duringthe growing season compared to the 2011-12 season, the weight of 20seeds from plants grown in the field was 0.977 g compared to 1.202 gfrom plants grown in the greenhouse. Eighteen to 20 seeds of each groupwere analysed for fatty acid composition by GC analysis. The oleic acidlevel in field grown seeds ranged from 74.83 to 80.65 with a mean (+/1s.d.) of 78.52+/−1.53, compared to a range in greenhouse grown seeds of75.15-78.44 with a mean of 76.33+/−1.00. Other fatty acids were presentat level in Table 20.

TABLE 20 Fatty acid composition of S317 seedoil grown in either thefield or the greenhouse. 18:3 18:2 Δ9z 16:1 18:1 18:1 Δ9z Δ12z 20:1Sample 16:0 Δ9z 18:0 Δ9z Δ11z Δ12z Δ15z 20:0 Δ11z 22:0 Field-grownAverage 4.90 0.00 2.40 78.52 0.75 12.5 0.00 0.47 0.20 0.21 Standard 0.240.00 0.31 1.53 0.28 1.66 0.00 0.05 0.13 0.13 deviation Greenhouse- grownAverage 4.80 0.04 2.72 76.33 0.72 14.2 0.00 0.46 0.23 0.25 Standard 0.10.04 0.3 1.00 0.17 1.06 0.00 0.12 0.06 0.06 deviation

Example 15. Crossing of Transgenes into Other Safflower Varieties

Safflower varieties are manually crossed using well-established methods,for example as described in Mündel and Bergman (2009). The best of thetransformed lines containing the constructs described above areselected, particularly transformed lines containing only a single T-DNAinsertion, and crossed with plants of other varieties of safflower,non-transformed or already transformed with a different construct, whichhave optimal agronomic performance. Using repeated rounds ofback-crossing with the recurrent parent, for example for 4 or 5backcrosses, and then selfing, plants are produced which are homozygousfor the desired construct(s) in the genetic background for optimalagronomic performance. Marker assisted selection may be used in thebreeding process, such as for example the use of a perfect marker forthe ol allele as described in Example 7.

Example 16. Modification of Seedoil Composition by ArtificialMicroRNA-Mediated Gene Silencing

MicroRNAs (miRNAs) are a class of 20-24-nucleotide (nt) regulatory smallRNAs (sRNA) endogenous to both plants and animals which regulateendogenous gene activity. Transgenic expression of modified miRNAprecursor RNAs (artificial miRNA precursors) represents a recentlydeveloped RNA-based and sequence specific strategy to silence endogenousgenes. It has been demonstrated that the substitution of severalnucleotides within the miRNA precursor sequence to make an artificialmiRNA precursor does not affect the biogenesis of the miRNA as long asthe positions of matches and mismatches within the precursor stem loopremain unaffected.

The CSIRO software package MatchPoint (www.pi.csiro.au/RNAi) (Horn andWaterhouse, 2010) was used to identify specific 21-mer sequences in theArabidopsis FAD2, FATB and FAE1 genes which were unique in theArabidopsis genome and therefore less likely to cause silencing ofnon-target genes (off-gene targeting) when expressed as artificialmiRNAs. A unique 21-mer sequence was selected for each of the 3 genes,the 21-mer in each case being fully complementary (antisense) to aregion of the transcript of the corresponding gene. An artificial miRNAprecursor molecule was designed for each, based on the A. thalianaara-miR159b precursor sequence. In each case, the miR159b precursorsequence was modified in its stem to accommodate the antisense 21-mersequence. Three constructs each encoding one of the precursor RNAs weremade, each under the control of the seed-specific FP1 promoter, andcloned into a binary expression vector to generate the constructsdesignated pJP1106, pJP1109 and pJP1110.

These constructs were separately transformed into A. tumefaciens strainAGL1 by electroporation and the transformed strains used to introducethe genetic construct into A. thaliana (ecotype Columbia) by the floraldipping method (Example 1). Seeds (Ti seeds) from the treated plantswere plated out on MS media supplemented with 3.5 mg/L PPT to selecttransformed seedlings, which were transferred to soil to establishconfirmed Ti transgenic plants. Most of these Ti plants were expected tobe heterozygous for the introduced genetic construct. T2 seed from thetransgenic plants were collected at maturity and analysed for theirfatty acid composition. These T2 plants included lines that werehomozygous for the genetic construct as well as ones which wereheterozygous. Homozygous T2 plants were self-fertilised to produce T3seed, and T3 progeny plants obtained from these seed in turn used toobtain T4 progeny plants. This therefore allowed the analysis of thestability of the gene silencing over three generations of progenyplants.

The fatty acid profiles of seedoil obtained from the T2, T3 and T4 seedlots were analysed by GC as described in Example 1. Alterations to theactivity of the Δ12-desaturase caused by the action of the FAD2-basedtransgene were seen as an increase in the amount of oleic acid in theseed oil profiles. A related method of assessing the cumulative effectsof Δ12-desaturase activity during seed fatty acid synthesis was throughcalculating the oleic desaturation proportion (ODP) parameter for eachseedoil, obtained by using the following formula:ODP=%18:2+%18:3/%18:1+%18:2+%18:3. Wild-type Arabidopsis seedoiltypically has an ODP value of around 0.70 to 0.79, meaning that 70% to79% of 18:1 formed during fatty acid synthesis in the seed wassubsequently converted to the polyunsaturated C18 fatty acids, first ofall by the action of Δ12-desaturase to produce 18:2 and then by furtherdesaturation to 18:3. The ODP parameter was therefore useful indetermining the extent of FAD2 gene-silencing on the level of endogenousΔ12-desaturase activity.

Levels of C18:1^(Δ9) (oleic acid) in T₂ seed transformed with thepJP1106 construct (FAD2 target) ranged from 32.9% to 62.7% in 30transgenic events compared to an average wild-type C18:1 level of14.0%±0.2. A highly silenced line (plant ID-30) which had a singletransgene insertion, determined by segregation ratios (3:1) of the plantselectable marker (PPT), was forwarded to the next generation (T3).Similarly high levels of the 18:1^(Δ9) were observed in T3 seed rangingfrom 46.0% to 63.8% with an average of 57.3+5.0%. In the followinggeneration, T4 seed also showed similarly high levels of 18:1^(Δ9)ranging from 61% to 65.8% with an average of 63.3+1.06%. The total PUFAcontent (18:2+18:3) in T2 transgenic seedoil ranged from 6.1% to 38%,but in the seedoil from the homozygous line ID-30, the total PUFAcontent was further reduced and ranged from 4.3 to 5.7%. The controlArabidopsis ecotype Columbia seedoil had an ODP value ranging from0.75-0.79, meaning that over 75% of oleic acid produced in thedeveloping seed was subsequently converted to 18:2 or 18:3. In contrast,the seedoil from the fad2-1 mutant of Arabidopsis had an ODP value of0.17, indicating about a 75% reduction in Δ12-desaturation due to thefad2-1 mutation. The ODP value ranged from 0.08 to 0.48 in the T2transgenic seedoil, 0.07-0.32 in the T3 seedoil and 0.06-0.08 in the T4seedoil, in contrast to the value of 0.75 in the control Arabidopsisseedoil. The drastic reduction in ODP values in the transgenic linesclearly indicated the efficient silencing of the endogenous FAD2 geneusing the artificial microRNA approach. This experiment also showed thestability of the gene silencing over three generations. Similar extentsof gene silencing were seen with the other two constructs todown-regulate their corresponding genes.

The degree of FAD2 gene silencing and the amount of 18:1^(Δ9)(63.3±1.06%) observed in this study using artificial microRNA was higherthan in the well characterised FAD2-2 mutant (59.4%), the FAD2 silencedline using the hairpin RNA approach (56.9±3.6%) and thehairpin-antisense approach (61.7±2.0%). The mean 18:2+18:3% content inFAD2 silenced seedoil using amiRNA was 4.8±0.37%, which was lower thanin the previously reported FAD2-2 mutant (7.5±1.1%) and the FAD2silenced line using the hairpin-antisense approach (7.2±1.4%). Thesedata therefore showed the advantages of the artificial microRNA in theextent of silencing, as well as the stability of silencing over thegenerations of progeny.

Example 17. Assaying Sterol Content and Composition in Oils

The phytosterols from 12 vegetable oil samples purchased from commercialsources in Australia were characterised by GC and GC-MS analysis asO-trimethylsilyl ether (OTMSi-ether) derivatives as described inExample 1. Sterols were identified by retention data, interpretation ofmass spectra and comparison with literature and laboratory standard massspectral data. The sterols were quantified by use of a5β(H)-Cholan-24-ol internal standard. The basic phytosterol structureand the chemical structures of some of the identified sterols are shownin FIG. 14 and Table 21.

TABLE 21 IUPAC/systematic names of identified sterols. Sterol No. Commonname(s) IUPAC/Systematic name 1 cholesterol cholest-5-en-3β-ol 2brassicasterol 24-methylcholesta-5,22E-dien-3β-ol 3 chalinasterol/24-24-methylcholesta-5,24(28)E-dien-3β-ol methylene cholesterol 4campesterol/24- 24-methylcholest-5-en-3β-ol methylcholesterol 5campestanol/24- 24-methylcholestan-3β-ol methylcholestanol 7Δ5-stigmasterol 24-ethylcholesta-5,22E-dien-3β-o l 9 ergost-7-en-3β-ol24-methylcholest-7-en-3β-ol 11 eburicol4,4,14-trimthylergosta-8,24(28)-dien- 3β-ol 12 β-sitosterol/24-24-ethylcholest-5-en-3β-ol ethylcholesterol 13 Δ5-24-ethylcholesta-5,24(28)Z-dien-3β-ol avenasterol/isofucosterol 19Δ7-stigmasterol/stigmast- 24-ethylcholest-7-en-3β-ol 7-en-3b-ol 20Δ7-avenasterol 24-ethylcholesta 7,24(28)-dien-3β-ol

The vegetable oils analysed were from: sesame (Sesamum indicum), olive(Olea europaea), sunflower (Helianthus annus), castor (Ricinuscommunis), canola (Brassica napus), safflower (Carthamus tinctorius),peanut (Arachis hypogaea), flax (Linum usitatissimum) and soybean(Glycine max). In decreasing relative abundance, across all of the oilsamples, the major phytosterols were: β-sitosterol (range 28-55% oftotal sterol content), Δ5-avenasterol (isofucosterol) (3-24%),campesterol (2-33%), □5-stigmasterol (0.7-18%), Δ7-stigmasterol (1-18%)and Δ7-avenasterol (0.1-5%). Several other minor sterols wereidentified, these were: cholesterol, brassicasterol, chalinasterol,campestanol and eburicol. Four C29:2 and two C30:2 sterols were alsodetected, but further research is required to complete identification ofthese minor components. In addition, several other unidentified sterolswere present in some of the oils but due to their very low abundance,the mass spectra were not intense enough to enable identification oftheir structures.

The sterol contents expressed as mg/g of oil in decreasing amount were:canola oil (6.8 mg/g), sesame oil (5.8 mg/g), flax oil (4.8-5.2 mg/g),sunflower oil (3.7-4.1 mg/g), peanut oil (3.2 mg/g), safflower oil (3.0mg/g), soybean oil (3.0 mg/g), olive oil (2.4 mg/g), castor oil (1.9mg/g). The % sterol compositions and total sterol content are presentedin Table 22.

Among all the seed oil samples, the major phytosterol was generallyβ-sitosterol (range 30-57% of total sterol content). There was a widerange amongst the oils in the proportions of the other major sterols:campesterol (2-17%), Δ5-stigmasterol (0.7-18%), Δ5-avenasterol (4-23%),Δ7-stigmasterol (1-18%). Oils from different species had a differentsterol profile with some having quite distinctive profiles. Canola oilhad the highest proportion of campesterol (33.6%), while the otherspecies samples generally had lower levels, e.g. up to 17% in peanutoil. Safflower oil had a relatively high proportion of Δ7-stigmasterol(18%), while this sterol was usually low in the other species oils, upto 9% in sunflower oil. Because they were distinctive for each species,sterol profiles can therefore be used to help in the identification ofspecific vegetable or plant oils and to check their genuineness oradulteration with other oils.

Two samples each of sunflower and safflower were compared, in each caseone was produced by cold pressing of seeds and unrefined, while theother was not cold-pressed and refined. Although some differences wereobserved, the two sources of oils had similar sterol compositions andtotal sterol contents, suggesting that processing and refining hadlittle effect on these two parameters. The sterol content among thesamples varied three-fold and ranged from 1.9 mg/g to 6.8 mg/g. Canolaoil had the highest and castor oil the lowest sterol content.

TABLE 22 Sterol content and composition of assayed plant oils. Sun- Saf-flower flower Sterol Sterol common Sun- cold- Saf- cold- Flax Flaxnumber* name Sesame Olive flower pressed Castor Canola flower pressedPeanut (linseed) (linseed) Soybean 1 cholesterol 0.2 0.8 0.2 0.0 0.1 0.30.2 0.1 0.2 0.4 0.4 0.2 2 brassicasterol 0.1 0.0 0.0 0.0 0.3 0.1 0.0 0.00.0 0.2 0.2 0.0 3 chalinasterol/24- 1.5 0.1 0.3 0.1 1.1 2.4 0.2 0.1 0.91.5 1.4 0.8 methylene cholesterol 4 campesterol/24- 16.2 2.4 7.4 7.9 8.433.6 12.1 8.5 17.4 15.7 14.4 16.9 methylcholesterol 5 campestanol/24-0.7 0.3 0.3 0.1 0.9 0.2 0.8 0.8 0.3 0.2 0.2 0.7 methylcholestanol 6C29:2* 0.0 0.0 0.1 0.2 0.0 0.1 0.5 0.5 0.0 1.2 1.3 0.1 7 Δ5-stigmasterol6.4 1.2 7.4 7.2 18.6 0.7 7.0 4.6 6.9 5.1 5.8 17.6 8 unknown 0.5 1.3 0.70.6 0.8 0.7 0.7 1.3 0.4 0.7 0.6 1.3 9 ergost-7-en-3β-ol 0.1 0.1 1.9 1.80.2 0.4 2.7 4.0 1.4 1.4 1.4 1.0 10 unknown 0.0 1.3 0.9 0.8 1.2 0.9 1.80.7 1.2 0.7 0.5 0.7 11 eburicol 1.6 1.8 4.1 4.4 1.5 1.0 1.9 2.9 1.2 3.53.3 0.9 12 β-sitosterol/24- 55.3 45.6 43.9 43.6 37.7 50.8 40.2 35.1 57.229.9 28.4 40.2 ethylcholesterol 13 Δ5-avenasterol/ 8.6 16.9 7.2 4.1 19.34.4 7.3 6.3 5.3 23.0 24.2 3.3 isofucosterol 14 triterpenoid 0.0 2.4 0.91.1 0.0 0.0 1.6 1.9 0.0 0.0 0.0 0.9 alcohol 15 triterpenoid 0.0 0.0 0.70.6 0.0 0.0 2.8 1.8 0.0 0.0 0.3 0.0 alcohol 16 C29:2* 0.0 0.5 0.7 0.71.5 1.2 2.8 1.9 2.0 1.0 0.7 0.5 17 C29:2* 1.0 0.9 2.3 2.4 0.6 0.4 1.31.9 0.9 1.0 1.0 1.0 18 C30:2* 0.0 0.0 0.0 0.0 1.9 0.0 0.0 0.0 0.0 0.00.0 0.0 19 Δ7-stigmasterol/ 2.2 7.1 9.3 10.9 2.3 0.9 10.5 18.3 1.1 7.98.7 5.6 stigmast-7-en- 3β-ol 20 Δ7-avenasterol 1.3 0.1 4.0 3.6 0.6 0.22.0 4.7 0.7 0.4 0.4 0.6 21 unknown 0.7 7.1 0.9 0.8 0.0 0.4 0.3 0.4 0.03.0 3.6 0.0 22 unknown 0.3 0.0 0.3 0.9 0.0 0.0 1.2 1.3 0.2 0.1 0.0 0.323 unknown 0.2 0.2 0.3 0.3 0.2 0.1 0.3 0.2 0.2 0.1 0.2 0.5 24 unknown0.0 3.1 0.9 1.3 0.6 0.4 0.2 0.4 0.7 1.7 1.9 0.8 25 unknown 0.9 0.4 0.30.5 0.3 0.1 0.5 0.7 0.3 0.1 0.1 0.6 26 C30:2 2.2 6.0 4.6 5.7 1.4 0.6 1.01.2 1.2 1.2 1.1 5.2 27 unknown 0.0 0.4 0.4 0.3 0.3 0.2 0.1 0.2 0.3 0.10.0 0.3 Sum 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0100.0 100.0 Total sterol 5.8 2.4 4.1 3.7 1.9 6.8 3.2 3.0 3.2 4.8 5.2 3.0(mg/g oil) C29:2* and and C30:2* denotes a C29 sterol with two doublebonds and a C30 sterol with two double bonds, respectively

A separate analysis was performed of safflower oils from green housederived control seed (S series), genetically modified high oleic acidseed (T series) and two commercial safflower oils. Several features wereobserved (Table 23). First, there is a high degree of similarity insterol pattern between the control and modified seeds and secondly thecommercial safflower oils are in a separate grouping and are thereforeshown to have significantly different phytosterol profile. Furtherexamination of the phytosterol profiles also showed the similarity ofthe phytosterol profiles from the control and modified safflower seedsamples.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

The present application claims priority from U.S. 61/638,447 filed 25Apr. 2012, and AU 2012903992 filed 11 Sep. 2012, both of which areincorporated herein by reference.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

TABLE 23 Sterol composition (% of total sterols) and content (mg/g) ofsafflower seed oil samples. P2589 P2590 P2591 P2592 P2593 Sterol S317S317 TS603.9.2 TS603.9.4 TS603.9.5 No* common name(s) IUPAC/systematicname (1) (2) T1 T1 T1 1 cholesterol cholest-5-en-3β-ol 0.5 0.6 0.5 3.01.1 2 brassicasterol 24-methylcholesta-5,22E-dien-3β-ol 0.0 0.0 0.1 0.00.1 3 chalinasterol/24-methylene cholesterol24-methylcholesta-5,24(28)E-dien-3β-ol 0.8 0.8 1.1 1.1 1.2 4campesterol/24-methylcholesterol 24-methylcholest-5-en-3β-ol 10.5 11.611.0 11.8 11.8 5 campestanol/24-methylcholestanol24-methylcholestan-3β-ol 0.0 0.1 0.2 0.0 0.3 6 C29:2** 0.9 0.8 0.2 7.20.4 7 Δ5-stigmasterol 24-ethylcholesta-5,22E-dien-3β-o l 0.7 0.9 0.8 0.80.6 8 unk*** 1.8 2.1 2.3 2.1 2.0 9 ergost-7-en-3β-ol24-methylcholest-7-en-3β-ol 2.8 3.3 2.6 2.3 2.6 10 unk*** 1.5 1.6 1.31.8 1.4 11 eburicol 4,4,14-trimthylergosta-8,24(28)-dien-3β-ol 1.7 2.25.0 2.8 2.6 12 β-sitosterol/24-ethylcholesterol24-ethylcholest-5-en-3β-ol 35.9 37.0 35.7 36.2 37.5 13Δ5-avenasterol/isofucosterol 24-ethylcholesta-5,24(28)Z-dien-3β-ol 10.48.2 10.0 7.9 9.3 14 triterpenoid alcohol 1.6 1.9 1.4 1.2 1.4 15triterpenoid alcohol 1.8 2.2 1.3 0.9 1.6 16 C29:2** 4.4 0.6 3.1 2.8 2.117 C29:2** 2.2 2.2 2.1 2.1 1.7 18 C30:2** 1.9 0.8 1.8 1.9 2.1 19Δ7-stigmasterol/stigmast-7-en-3β-ol 24-ethylcholest-7-en-3β-ol 11.4 13.610.3 8.9 10.7 20 Δ7-avenasterol**** 24-ethylcholesta7,24(28)Z-dien-3β-ol 6.1 5.7 5.3 3.4 4.9 21 unk*** 0.2 0.4 0.2 0.5 0.422 unk*** 0.9 1.0 1.1 1.1 0.9 23 unk*** 0.0 0.0 0.0 0.0 0.0 24 unk***0.2 0.6 0.9 1.5 0.9 25 unk*** 0.9 0.6 0.3 1.0 0.5 26 C30:2 0.9 1.0 0.52.9 1.4 27 unk*** 0.0 0.1 0.8 2.2 0.4 Sum 100.0 100.0 100.0 100.0 100.0Total sterol (mg/g oil) 1.9 2.2 2.0 2.0 1.8 *Sterol numbers refer to GCtraces. **C29:2 and and C30:2 denotes C29 sterol with two double bondsand C30 sterol with two double bonds, respectively. ***unk denotesunknown. ****tentative identification. S317 samples are unmodifiedparental controls and TS samples are high oleic acid modified saffloweroil samples

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The invention claimed is:
 1. A safflower seed comprising (a) a firstexogenous polynucleotide which encodes a first silencing RNA which is adouble-stranded RNA (dsRNA) molecule or a microRNA comprising a sequencewhich is complementary to a region of a mRNA of a CtFAD2-2 gene andwhich is capable of reducing expression of the CtFAD2-2 gene in adeveloping safflower seed relative to a corresponding safflower seedlacking the exogenous polynucleotide, and wherein the first exogenouspolynucleotide is operably linked to a promoter which directs expressionof the polynucleotide in the developing safflower seed, and (b) a secondexogenous polynucleotide which encodes a second silencing RNA which is adouble-stranded RNA (dsRNA) molecule or a microRNA comprising a sequencewhich is complementary to a region of a mRNA of a CtFATB-3 gene andwhich is capable of reducing expression of the CtFATB-3 gene in adeveloping oilseed or safflower seed relative to a corresponding oilseedor safflower seed lacking the second exogenous polynucleotide, andwherein the second exogenous polynucleotide is operably linked to apromoter which directs expression of the polynucleotide in thedeveloping oilseed or safflower seed, wherein: (i) the first silencingRNA reduces the expression of both CtFAD2-1 and CtFAD2-2 genes; or (ii)the safflower seed comprises a homozygous mutation in a CtFAD2-1 gene,wherein the mutation reduces the activity of the CtFAD2-1 gene indeveloping safflower seed relative to a corresponding safflower seedlacking the mutation, and wherein 90% to 95% by weight of the totalfatty acids of the oil content of the safflower seed is oleic acid.
 2. Asafflower plant which produces the safflower seed of claim
 1. 3. Thesafflower seed according to claim 1 which comprises a third exogenouspolynucleotide which encodes a third silencing RNA which is adouble-stranded RNA (dsRNA) molecule or a microRNA comprising a sequencewhich is complementary to a region of a mRNA of a plastidial ω6 fattyacid desaturase (FAD6) gene and which is capable of reducing expressionof the FAD6 gene in a developing safflower seed relative to acorresponding safflower seed lacking the third exogenous polynucleotide,and wherein the third exogenous polynucleotide is operably linked to apromoter which directs expression of the polynucleotide in thedeveloping safflower seed.
 4. The safflower seed according to claim 1,wherein the first exogenous polynucleotide and the second exogenouspolynucleotide are covalently joined on a single DNA molecule,optionally with a linking DNA sequence between the first and secondexogenous polynucleotides.
 5. The safflower seed of claim 4, wherein thefirst exogenous polynucleotide and the second exogenous polynucleotideare under the control of a single promoter such that, when the firstexogenous polynucleotide and the second exogenous polynucleotide aretranscribed in the developing safflower seed, the first silencing RNAand the second silencing RNA are covalently linked as parts of a singleRNA transcript.
 6. The safflower seed according to claim 1, whichcomprises a single transfer DNA integrated into the genome of thesafflower seed, and wherein the single transfer DNA comprises the firstexogenous polynucleotide and the second exogenous polynucleotide.
 7. Thesafflower seed of claim 6 which is homozygous for the transfer DNA. 8.The safflower seed according to claim 1, wherein all of the promotersare seed specific and preferentially expressed in the embryo of adeveloping safflower seed.
 9. The safflower seed of claim 1, whichcomprises a homozygous mutation in a CtFAD2-1 gene wherein the mutationis selected from a deletion, an insertion, an inversion, a frameshift, apremature translation stop codon, or one or more non-conservative aminoacid substitutions.
 10. The safflower seed of claim 9, wherein themutation is a null mutation in the CtFAD2-1 gene.
 11. The safflower seedaccording to claim 1 comprising an ol allele of the CtFAD2-1 gene or anoil allele of the CtFAD2-1 gene, or both alleles.
 12. The safflower seedaccording to claim 1 wherein the first silencing RNA reduces theexpression of both CtFAD2-1 and CtFAD2-2 genes.
 13. A safflower plantwhich produces the safflower seed of claim
 3. 14. The safflower plant ofclaim 2 which is transgenic and homozygous for an insertion into itsgenome which comprises the first exogenous polynucleotide and the secondexogenous polynucleotide.
 15. The safflower seed of claim 1, wherein (a)the first silencing RNA comprises at least 19 consecutive nucleotidescomplementary to a region of SEQ ID NO:2; and (b) the second silencingRNA comprises at least 19 consecutive nucleotides complementary to aregion of SEQ ID NO:41.